Amino acid sequences directed against HER2 and polypeptides comprising the same for the treatment of cancers and/or tumors

ABSTRACT

The present invention relates to amino acid sequences and Nanobodies that are directed against Epidermal Growth Factor Receptor 2 (HER2), as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 of international application PCT/EP2008/066363, filed Nov. 27, 2008, which was published under PCT Article 21(2) in English, and claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No. 60/004,332, filed Nov. 27, 2007, U.S. provisional application Ser. No. 60/005,265, filed Dec. 4, 2007, U.S. provisional application Ser. No. 60/005,324, filed Dec. 4, 2007, and U.S. provisional application Ser. No. 60/005,331, filed Dec. 4, 2007, the disclosures of which are incorporated by reference herein in their entireties.

The present invention relates to amino acid sequences that are directed against (as defined herein) Epidermal Growth Factor Receptor 2 (HER2), as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences (also referred to herein as “amino acid sequences of the invention”, “compounds of the invention”, and “polypeptides of the invention”, respectively).

The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to herein as “nucleic acids of the invention” or “nucleotide sequences of the invention”); to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.

Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.

HER2 (also known as HER-2, Her-2, ErbB-2, ERBB2, EGF receptor 2, HER2/neu) is a member of the ErbB protein family, also known as the ERBB or the epidermal growth factor receptor family. This subclass I of the receptor tyrosine kinase (RTK) superfamily comprises four members: EGFR/ERBB1, HER2/ERBB2, HER3/ERBB3 and HER4/ERBB4. All members have an extracellular ligand-binding region, a single membrane-spanning region and a cytoplasmic tyrosine-kinase-containing domain. The ERBB receptors are expressed in various tissues of epithelial, mesenchymal and neuronal origin. Under normal physiological conditions, activation of the ERBB receptors is controlled by the spatial and temporal expression of their ligands, which are members of the EGF family of growth factors (Riese and Stern, 1998, Bioessays 20: 41; Yarden and Sliwkowski, 2001, Nature Rev. Mol. Cell. Biol. 2: 127). Ligand binding to ERBB receptors induces the formation of receptor homo- and heterodimers and activation of the intrinsic kinase domain, resulting in phosphorylation on specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for a range of proteins, the recruitment of which leads to the activation of intracellular signalling pathways (Yarden and Sliwkowski, 2001, Nature Rev. Mol. Cell. Biol. 2: 127; Olayioye et al. 2000, EMBO J. 19: 3159; Schlessinger, 2004, Science 306: 1506; Hynes and Lane, 2005, Nature Reviews/Cancer 5: 341).

For the amino acid sequence of HER-2, reference is made to the sequences mentioned under Genbank accession numbers NM 001005862 en NM 004448 (both incorporated herein by reference). For the domain(s) of HER-2 involved in the interaction between HER-2 and Omnitarg and the amino acid sequence(s) thereof, reference is made to Franklin et al. (2004, Cancer cell 5:317-328; also incorporated herein by reference). For the domains of HER-2 involved in the interaction between HER-2 and Herceptin® and the amino acid sequence(s) thereof, reference is made to Cho et al. (2003, Nature 421:756-760; also incorporated herein by reference).

HER2 is thought to be an orphan receptor, with none of the EGF family of ligands able to activate it. However, ErbB receptors dimerise on ligand binding, and HER2 is the preferential dimerisation partner of other members of the ErbB family (Graus-Porta et al, 1997, EMBO J. 16: 1647).

The extracellular region of each ERBB receptor consists of four domains (I-IV). The structure of HER2's extracellular region is radically different from the other EGF receptors. In the other EGF receptors, in non-activated state, domain II binds to domain IV. Upon binding to domains I and III, the activating growth factor (ligand) selects and stabilizes a conformation that allows a dimerization arm to extend from domain II to interact with an ERBB dimer partner. HER2, on the other hand, has a fixed conformation that resembles the ligand-activated state: the domain II-IV interaction is absent and the dimerization loop in domain II is continuously exposed (in detail discussed in Hynes and Lane, 2005, Nature Reviews/Cancer 5: 341, Garrett et al. 2003, Mol. Cell. 11: 495; Cho et al. 2003, Nature 421: 756). This also explains why HER2 is the preferred dimerization partner.

Amplification of HER-2 leading to overexpression of the receptor, originally detected in a subset of breast tumours, occurs in various human cancers including ovarian, stomach, bladder, gastric and salivary cancers (Holbro and Hynes, 2004, Annu. Rev. Pharmacol. Toxicol. 44:195; Hynes and Stern, 1994, Biochim. Biophys. Acta 1198: 165). Approximately 25-30 percent of breast cancers have an amplification of the HER2/neu gene or overexpression of its protein product. Overexpression of this receptor in breast cancer is associated with increased disease recurrence and worse prognosis. Therefore, ERBB receptors have been intensely pursued as therapeutic targets (Holbro and Hynes, 2004, Annu. Rev. Pharmacol. Toxicol. 44:195).

mAb4D5, isolated by Ullrich et al. (Mol. Cell. Biol. 1989, 9: 1165), and trastuzumab (marketed as Herceptin®), its humanized (human IgG1 backbone, murine complementary-determining regions) variant (Carter et al. 1992, Proc. Natl. Acad. Sci. USA 89: 4285), block proliferation of HER2-overexpressing breast cancer cells. The structure of the trastuzumab Fab fragment bound to the extracellular portion of HER2 indicates that its epitope is toward the carboxyterminus of domain IV (Cho et al. 2003, Nature 421: 756). Domain IV does not participate in receptor dimerization, and blockade of dimerization does not explain the mechanism of action of this antibody. The mechanisms underlying trastuzumab's clinical efficacy is still under debate and seems to be multifaceted. Its inherent ability to recruit immune effector cells such as macrophages and monocytes to the tumor through the binding of its constant Fc domain to specific receptors on these cells, might be relevant for its anti-tumor activity. In addition to this Fc-mediated functions, preclinical studies have shown that the antibody downregulates HER2 levels (Hudziak et al. 1989, Mol. Cell. Biol 9: 1165) and HER2-mediated signaling pathways (Lane et al. 2000, Mol. Cell. Biol. 20: 3210, Motoyama et al. 2002, Cancer Res. 62: 3151). Furthermore, metalloproteinase-mediated. HER2 ectodomain shedding has been proposed to cause constitutive HER2 signaling and trastuzumab also blocks this process (Molina et al. 2001, Cancer Res. 61: 4744). Trastuzumab is only effective in breast cancer where the HER2/neu receptor is overexpressed. Clinical trials showed that the addition of trastuzumab to standard chemotherapy prolonged relapse-free survival, leading to the approval of the drug for treatment of HER2-overexpressing metastatic breast cancer patients.

Another monoclonal antibody, pertuzumab (Omnitarg) (Olayioye, 2001, Breast Cancer Res 3: 385), which inhibits ligand activation of an ErbB hetero-oligomer comprising HER2 and HER3, HER4 or EGFR, is in advanced clinical trials. Pertuzumab binds to HER2 near the center of domain II. Binding is predicted to sterically block the region necessary for HER2 dimerization with other ERBBs (Franklin et al. 2004, Cancer Cell 5: 317), Pertuzumab but not trastuzumab inhibits the growth of tumors displaying low HER2 levels (Agus et al. 2002, Cancer Cell 2: 127).

A specific, but non-limiting object of the invention is to provide therapeutic compounds that have improved therapeutic and/or pharmacological properties and/or other advantageous properties (such as, for example, improved ease of preparation and/or reduced costs of goods), compared to these conventional antibodies. These improved and advantageous properties will become clear from the further description herein. The therapeutic compounds provided by the invention may, for example, have an increased avidity and/or potency, an increased selectivity and/or they may be capable of partially or totally blocking certain (one or more) sites.

The polypeptides and compositions of the present invention can generally be used to bind HER2 and, by this binding to HER2, modulate, and in particular inhibit or prevent, the signalling that is mediated by HER2, to modulate the biological pathways in which HER2 is involved, and/or to modulate the biological mechanisms, responses and effects associated with such signalling or these pathways (which are also referred to herein as “modes of action” of the polypeptides and compositions of the invention).

One specific, non-limiting, object of the invention is to provide therapeutic compounds that combine two or more modes of action, e.g. by blocking of two or more different cell signalling pathways. One specific, but non-limiting object of the invention is to provide therapeutic compounds that combine the mode of action of Herceptin® and Omnitarg.

The polypeptides and compositions of the present invention can be used to modulate, and in particular inhibit and/or prevent, dimerization of HER2 with an ERBB receptor, and thus to modulate, and in particular inhibit or prevent, the signalling that is mediated by dimerization of HER2 with said ERBB receptor, to modulate the biological pathways in which HER2 and/or said ERBB receptor are involved, and/or to modulate the biological mechanisms, responses and effects associated with such signalling or these pathways.

As such, the polypeptides and compositions of the present invention can be used for the prevention and treatment (as defined herein) of cancers and/or tumors. Generally, “cancers and/or tumors” can be defined as diseases and disorders that can be prevented and/or treated, respectively, by suitably administering to a subject in need thereof (i.e. having the disease or disorder or at least one symptom thereof and/or at risk of attracting or developing the disease or disorder) of either a polypeptide or composition of the invention (and in particular, of a pharmaceutically active amount thereof) and/or of a known active principle active against HER2 or a biological pathway or mechanism in which HER2 is involved (and in particular, of a pharmaceutically active amount thereof). Examples of such cancers and/or tumors will be clear to the skilled person based on the disclosure herein, and for example include the following diseases and disorders: breast cancer and/or tumors, ovarian cancer and/or tumors, stomach cancer and/or tumors, bladder cancer and/or tumors, gastric cancer and/or tumors, salivary cancer and/or tumors, and prostate cancer.

In particular, the polypeptides and compositions of the present invention can be used for the prevention and treatment of cancers and/or tumors which are characterized by excessive and/or unwanted signalling mediated by HER2 or by the pathway(s) in which HER2 is involved. Examples of such cancers and/or tumors will again be clear to the skilled person based on the disclosure herein.

Thus, without being limited thereto, the amino acid sequences and polypeptides of the invention can for example be used to prevent and/or to treat all diseases and disorders that are currently being prevented or treated with active principles that can modulate HER2-mediated signalling, such as those mentioned in the prior art cited above. It is also envisaged that the polypeptides of the invention can be used to prevent and/or to treat all diseases and disorders for which treatment with such active principles is currently being developed, has been proposed, or will be proposed or developed in future. In addition, it is envisaged that, because of their favourable properties as further described herein, the polypeptides of the present invention may be used for the prevention and treatment of other diseases and disorders than those for which these known active principles are being used or will be proposed or developed; and/or that the polypeptides of the present invention may provide new methods and regimens for treating the diseases and disorders described herein.

Other applications and uses of the amino acid sequences and polypeptides of the invention will become clear to the skilled person from the further disclosure herein.

Generally, it is an object of the invention to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the diagnosis, prevention and/or treatment of cancers and/or tumors and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or use of such agents and compositions.

In particular, it is an object of the invention to provide such pharmacologically active agents, compositions and/or methods that have certain advantages compared to the agents, compositions and/or methods that are currently used and/or known in the art. These advantages will become clear from the further description below.

More in particular, it is an object of the invention to provide therapeutic proteins that can be used as pharmacologically active agents, as well as compositions comprising the same, for the diagnosis, prevention and/or treatment of cancers and/or tumors and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or the use of such therapeutic proteins and compositions.

Accordingly, it is a specific object of the present invention to provide amino acid sequences that are directed against (as defined herein) HER2, in particular against HER2 from a warm-blooded animal, more in particular against HER2 from a mammal, and especially against human HER2 (and specifically, against human HER-2 with the amino acid sequence given under Genbank accession numbers NM 001005862 en NM 004448); and to provide proteins and polypeptides comprising or essentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that are suitable for prophylactic, therapeutic and/or diagnostic use in a warm-blooded animal, and in particular in a mammal, and more in particular in a human being.

More in particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used for the prevention, treatment, alleviation and/or diagnosis of one or more diseases, disorders or conditions associated with HER2 and/or mediated by HER2 (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

It is also a specific object of the invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used in the preparation of pharmaceutical or veterinary compositions for the prevention and/or treatment of one or more diseases, disorders or conditions associated with and/or mediated by HER2 (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

In the invention, generally, these objects are achieved by the use of the amino acid sequences, proteins, polypeptides and compositions that are described herein.

In general, the invention provides amino acid sequences that are directed against (as defined herein) and/or can specifically bind (as defined herein) to HER2; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

More in particular, the invention provides amino acid sequences that can bind to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the invention are preferably such that they:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to         10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²         moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²         moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to         10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles         or more and more preferably 10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about         10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,         more preferably between 10⁴M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as         between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;         and/or such that they:     -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)         and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a         t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶         s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as         between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or a polypeptide that contains only one amino acid sequence of the invention) is preferably such that it will bind to HER2 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

Some preferred IC₅₀ values for binding of the amino acid sequences or polypeptides of the invention to HER2 will become clear from the further description and examples herein.

For binding to HER2, an amino acid sequence of the invention will usually contain within its amino acid sequence one or more amino acid residues or one or more stretches of amino acid residues (i.e. with each “stretch” comprising two or more amino acid residues that are adjacent to each other or in close proximity to each other, i.e. in the primary or tertiary structure of the amino acid sequence) via which the amino acid sequence of the invention can bind to HER2, which amino acid residues or stretches of amino acid residues thus form the “site” for binding to HER2 (also referred to herein as the “antigen binding site”).

The amino acid sequences provided by the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more amino acid sequences of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than HER2), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. Such a protein or polypeptide may also be in essentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as such preferably essentially consist of a single amino acid chain that is not linked via disulphide bridges to any other amino acid sequence or chain (but that may or may not contain one or more intramolecular disulphide bridges. For example, it is known that Nanobodies—as described herein—may sometimes contain a disulphide bridge between CDR3 and CDR1 or FR2). However, it should be noted that one or more amino acid sequences of the invention may be linked to each other and/or to other amino acid sequences (e.g. via disulphide bridges) to provide peptide constructs that may also be useful in the invention (for example Fab′ fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and other multispecific constructs. Reference is for example made to the review by Holliger and Hudson, Nat Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound, construct or polypeptide comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably either an amino acid sequence that does not occur naturally in said subject; or, when it does occur naturally in said subject, in essentially isolated form (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use, the amino acid sequences of the invention (as well as compounds, constructs and polypeptides comprising the same) are preferably directed against human HER2; whereas for veterinary purposes, the amino acid sequences and polypeptides of the invention are preferably directed against HER2 from the species to be treated, or at least cross-reactive with HER2 from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, and in addition to the at least one binding site for binding against HER2, contain one or more further binding sites for binding against other antigens, proteins or targets. The efficacy of the amino acid sequences and polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person, and for example include BIAcore binding assay, FACS binding and/or competition assay, ELISA binding and/or competition assay, FMAT binding and/or competition assay, Alphascreen binding and/or competition assay, tumor (e.g. SKBR3) cell proliferation assay (Hudziak et al., Molecular and Cellular Biology 9:1165-1172, 1989), cell signalling assays (Agus et al., Cancer Cell 2:127-136, 2002), SCID mice with implanted tumor (i.e. Xenograft mice) (Agus et al., Cancer Cell 2:127-136, 2002), HER2-transgenic mice (Scwall et al., Breast Cancer Res 5(Suppl 1):14, 2003), as well as the assays and animal models used in the experimental part below and in the prior art cited herein.

Also, according to the invention, amino acid sequences and polypeptides that are directed against HER2 from a first species of warm-blooded animal may or may not show cross-reactivity with HER2 from one or more other species of warm-blooded animal. For example, amino acid sequences and polypeptides directed against human HER2 may or may not show cross reactivity with HER2 from one or more other species of primates (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) and/or with HER2 from one or more species of animals that are often used in animal models for diseases (for example mouse, rat, rabbit, pig or dog), and in particular in animal models for diseases and disorders associated with HER2 (such as the species and animal models mentioned herein). In this respect, it will be clear to the skilled person that such cross-reactivity, when present, may have advantages from a drug development point of view, since it allows the amino acid sequences and polypeptides against human. HER2 to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the invention that are cross-reactive with HER2 from multiple species of mammal will usually be advantageous for use in veterinary applications, since it will allow the same amino acid sequence or polypeptide to be used across multiple species. Thus, it is also encompassed within the scope of the invention that amino acid sequences and polypeptides directed against HER2 from one species of animal (such as amino acid sequences and polypeptides against human HER2) can be used in the treatment of another species of animal, as long as the use of the amino acid sequences and/or polypeptides provide the desired effects in the species to be treated.

The present invention is in its broadest sense also not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain (I, II, III and/or IV), subunit or conformation (where applicable) of HER2 against which the amino acid sequences and polypeptides of the invention are directed. For example, the amino acid sequences and polypeptides may or may not be directed against an “interaction site” (as defined herein). However, it is generally assumed and preferred that the amino acid sequences and polypeptides of the invention are preferably at least directed against an interaction site (as defined herein), and in particular against the Herceptin® binding site on HER2 (see Cho et al, (2003), Nature 421:756-760), the Omnitarg binding site on HER2 (see Franklin et al. (2004), Cancer cell 5:317-328), or the Herceptin® binding site and the Omnitarg binding site on HER2.

An amino acid of the invention that is directed against and/or binds one specific antigenic determinant, or epitope of a target or antigen (such as a specific antigenic determinant, epitope, part, domain (I, II, III and/or IV) or subunit of HER2) while not binding any other antigenic determinant, or epitope of the target or antigen and not binding any other target or antigen, is also referred to herein as monovalent amino acid or monovalent construct of the invention.

As further described herein, a polypeptide of the invention may contain two or more (monovalent) amino acid sequences or monovalent constructs of the invention that are directed against HER2. Generally, such polypeptides will bind to HER2 with increased avidity compared to a single amino acid sequence of the invention. Such a polypeptide may for example comprise two amino acid sequences of the invention that are directed against the same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of HER2 (which may or may not be an interaction site); or comprise at least one “first” amino acid sequence of the invention that is directed against a first same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of HER2 (which may or may not be an interaction site); and at least one “second” amino acid sequence of the invention that is directed against a second antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) different from the first (and which again may or may not be an interaction site). Preferably, in such “biparatopic” polypeptides of the invention, at least one amino acid sequence of the invention is directed against an interaction site (as defined herein), although the invention in its broadest sense is not limited thereto.

Also, when the target is part of a binding pair (for example, a receptor-ligand binding pair), the amino acid sequences and polypeptides may be such that they compete with the cognate binding partner (e.g. the ligand, receptor or other binding partner, as applicable) for binding to the target, and/or such that they (fully or partially) neutralize binding of the binding partner to the target.

Thus, in one preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are directed against the Herceptin® binding site on HER2 and/or are capable of competing with Herceptin® for binding to HER-2, as determined using a suitable competition assay, such as the assay described in. Example 8. Such amino acid sequences and polypeptides of the invention may be as further defined herein. The amino acid sequences and polypeptides of the invention may in particular be directed against domain IV of HER2. In a preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the C-terminus of domain IV of HER2.

In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are capable, upon binding to HER-2, to (i) recruit immune effector cells such as macrophages and monocytes to the tumor (for this purpose, most preferably a polypeptide of the invention is used that contains an Fc portion that confers upon the polypeptide the ability to recruit immune effector cells such as macrophages and monocytes to the tumor); and/or (ii) modulate (as defined herein) HER-2 or HER-2 mediated signalling by downregulating HER2 levels (for example, as determined by the methodology described in Hudziak et al. 1989, Mol. Cell. Biol 9: 1165) and/or by downregulating HER2-mediated signaling pathways (for example, as determined by the methodology described in Lane et al. 2000, Mol. Cell. Biol. 20: 3210, Motoyama et al. 2002, Cancer Res. 62: 3151); and/or (iii) modulate (as defined herein) HER-2 or HER-2 mediated signalling by blocking or inhibiting metalloproteinase-mediated HER2 ectodomain shedding (for example, as determined by the methodology described in Molina et al. 2001, Cancer Res. 61: 4744); or more generally capable of modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Herceptin®. Such amino acid sequences and polypeptides of the invention preferably are directed against the Herceptin® binding site on HER2 and/or capable of competing with Herceptin® for binding to HER-2, and may in particular be directed against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2 (see also Cho et al. (2003), Nature 421:756-760).

In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are directed against the Omnitarg binding site on HER2 and/or are capable of competing with Omnitarg (and/or with the Omnitarg-Fab used in Example 9) for binding to HER-2, as determined using a suitable competition assay, such as the assay described in Example 9. Such amino acid sequences and polypeptides of the invention may be as further defined herein. The amino acid sequences and polypeptides of the invention may be directed against domain II of HER2. In a preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the center of domain II of HER2.

In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are capable, upon binding to HER-2, to modulate (as defined herein) HER-2 or HER-2 mediated signalling by inhibiting ligand activation of an ErbB hetero-oligomer comprising HER2 and HER3, HERO or EGFR, or more generally capable of modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Omnitarg. Such amino acid sequences and polypeptides of the invention preferably are directed against the Omnitarg binding site on HER2 and/or capable of competing with Omnitarg (and/or with the Omnitarg-Fab used in Example 9) for binding to HER-2, and may in particular be directed against domain II of HER2, and more in particular against the middle of domain II of HER2 (see also Franklin et al. (2004), Cancer cell 5:317-328).

It is also within the scope of the invention that, where applicable, an amino acid sequence or polypeptide of the invention can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or confirmations of HER2. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of HER2 to which the amino acid sequences and/or polypeptides of the invention bind may be essentially the same (for example, if HER2 contains repeated structural motifs or occurs in a multimeric form) or may be different (and in the latter case, the amino acid sequences and polypeptides of the invention may bind to such different antigenic determinants, epitopes, parts, domains, subunits of HER2 with an affinity and/or specificity which may be the same or different).

In a preferred aspect, the amino acid sequences and (in particular) polypeptides of the invention are capable of binding to two or more different antigenic determinants, epitopes, parts, domains of HER2. In this context, the amino acid sequences and polypeptides of the invention are also referred to as “multiparatopic” (such as e.g. “biparatopic” or “triparatopic”, etc.) amino acid sequences and polypeptides. The multiparatopic amino acid sequences and polypeptides of the invention can be directed against any antigenic determinants, epitopes, parts, and/or domains of HER2.

For example, and generally, a biparatopic polypeptide of the invention may comprise at least one amino acid sequence of the invention directed against a first antigenic determinant, epitope, part or domain of HER-2 and at least one amino acid sequence of the invention directed against a second antigenic determinant, epitope, part or domain of HER-2 different from the first antigenic determinant, epitope, part or domain (in which said amino acid sequences may be suitably linked, for example via a suitable linker as further described herein). Preferably, such a biparatopic polypeptide of the invention is further such that, when it binds to HER-2, it is capable of simultaneously binding to the first antigenic determinant, epitope, part or domain (i.e. via the at least one amino acid sequence of the invention capable of binding to said first antigenic determinant, epitope, part or domain) and binding to said second antigenic determinant, epitope, part or domain (i.e. via the at least one amino acid sequence of the invention capable of binding to said second antigenic determinant, epitope, part or domain). Examples of such biparatopic polypeptides of the invention will become clear from the further description herein. Also; a triparatopic polypeptide of the invention may comprise at least one further amino acid sequence of the invention directed against a third antigenic determinant, epitope, part or domain of HER-2 (different from both the first and second antigenic determinant, epitope, part or domain), and generally multiparatopic polypeptides of the invention may contain at least two amino acid sequences of the invention directed against at least two different antigenic determinants, epitopes, parts or domains of HER-2. Generally, such biparatopic, triparatopic and multiparatopic polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic, triparatopic and multiparatopic polypeptides of the invention (for example, these biparatopic, triparatopic and multiparatopic polypeptides of the invention preferably comprise single variable domains and more preferably Nanobodies).

In a preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are directed against the Herceptin® binding site on HER2 and/or capable of competing with Herceptin® for binding to HER-2, as well as against at least one other antigenic determinant, epitope, part or domain on HER2. The amino acid sequences and polypeptides of the invention may be directed against domain IV of HER2 as well as against at least one other antigenic determinant, epitope, part or domain on HER2. In a preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the C-terminus of domain IV of HER2 as well as against at least one other antigenic determinant, epitope, part or domain on. HER2. Generally, such a biparatopic (or multiparatopic) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the Herceptin® binding site on HER2 and/or capable of competing with Herceptin® for binding to HER-2 (and in particular against the domain IV of HER-2 and more preferably against the C-terminus of domain IV of HER2), as well as at least one further amino acid sequence of the invention that is capable of binding to at least one other antigenic determinant, epitope, part or domain on HER2. Generally, such biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the Herceptin® binding site and the at least one other antigenic determinant, epitope, part or domain on HER2; and preferably comprise single variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are at least capable, upon binding to HER-2, to (i) recruit immune effector cells such as macrophages and monocytes to the tumor (for this purpose, most preferably a polypeptide of the invention is used that contains an Fc portion that confers upon the polypeptide the ability to recruit immune effector cells such as macrophages and monocytes to the tumor); and/or (ii) modulate (as defined herein) HER-2 or HER-2 mediated signalling by downregulating HER2 levels (for example, as determined by the methodology described in Hudziak et al. 1989, Mol. Cell. Biol 9: 1165) and/or by downregulating HER2-mediated signaling pathways (for example, as determined by the methodology described in Lane et al. 2000, Mol. Cell. Biol. 20: 3210, Motoyama et al. 2002, Cancer Res. 62: 3151); and/or (iii) modulate (as defined herein) HER-2 or HER-2 mediated signalling by blocking or inhibiting metalloproteinase-mediated HER2 ectodomain shedding (for example, as determined by the methodology described in Molina et al. 2001, Cancer Res. 61: 4744); or more generally by modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Herceptin®.

Such biparatopic (or multiparatopic) polypeptides of the invention preferably either (a) comprise an Fc portion that confers upon the polypeptide the ability to recruit immune effector cells such as macrophages and monocytes to the tumor), and/or (b) comprise at least one amino acid sequence of the invention that is capable, upon binding to HER-2, to (1) modulate (as defined herein) HER-2 or HER-2 mediated signalling by downregulating HER2 levels and/or by downregulating HER2-mediated signaling pathways; and/or (2) modulate (as defined herein) HER-2 or HER-2 mediated signalling by blocking or inhibiting metalloproteinase-mediated HER2 ectodomain shedding; or more generally (3) modulate (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Herceptin®; as well as at least one further amino acid sequence of the invention that is capable of binding to at least one other antigenic determinant, epitope, part or domain on HER2 (i.e. different from the antigenic determinant, epitope, part or domain to which the aforementioned amino acid sequence of the invention can bind). Such biparatopic (or multiparatopic) polypeptides of the invention preferably comprise at least one amino acid sequence of the invention that is directed against the Herceptin® binding site on HER2 and/or capable of competing with Herceptin® for binding to HER-2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2), as well as at least one further amino acid sequence of the invention that is capable of binding to at least one other antigenic determinant, epitope, part or domain on HER2. Generally, such biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the Herceptin® binding site and the at least one other antigenic determinant, epitope, part or domain on HER2; and preferably comprise single variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are biparatopic (or multiparatopic) and are directed against the Omnitarg binding site on HER2 and/or capable of competing with Omnitarg for binding to HER-2, as well as against at least one other antigenic determinant on HER2. The amino acid sequences and polypeptides of the invention may be directed against domain II of HER2 as well as against at least one other antigenic determinant on HER2. In a preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the center of domain II of HER2 as well as against at least one other antigenic determinant on HER2. Generally, such a biparatopic (or multiparatopic) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the Omnitarg binding site on HER2 and/or capable of competing with Omnitarg for binding to HER-2 (and in particular against the domain II of HER-2 and more preferably against the middle of domain. II of HER2), as well as at least one further amino acid sequence of the invention that is capable of binding to at least one other antigenic determinant, epitope, part or domain on HER2. Generally, such biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the Omnitarg binding site and the at least one other antigenic determinant, epitope, part or domain on HER2; and preferably comprise single variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are at least capable, upon binding to HER-2, to modulate (as defined herein) HER-2 or HER-2 mediated signalling by inhibiting ligand activation of an ErbB hetero-oligomer comprising HER2 and HER3, HER4 or EGFR, or more generally capable of modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Omnitarg.

Such biparatopic (or multiparatopic) polypeptides of the invention preferably comprise at least one amino acid sequence of the invention that is capable, upon binding to HER-2, to modulate (as defined herein) HER-2 or HER-2 mediated signalling by inhibiting ligand activation of an ErbB hetero-oligomer comprising HER2 and HER3, HER4 or EGFR, or more generally capable of modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Omnitarg; as well as at least one further amino acid sequence of the invention that is capable of binding to at least one other antigenic determinant, epitope, part or domain on HER2 (i.e. different from the antigenic determinant, epitope, part or domain to which the aforementioned amino acid sequence of the invention can bind). Such biparatopic (or multiparatopic) polypeptides of the invention preferably comprise at least one amino acid sequence of the invention that is directed against the Omnitarg binding site on HER2 and/or capable of competing with Omnitarg for binding to HER-2 (and in particular against domain II of HER2, and more in particular against the middle of domain II of HER2), as well as at least one further amino acid sequence of the invention that is capable of binding to at least one other antigenic determinant, epitope, part or domain on HER2. Generally, such biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the Omnitarg binding site and the at least one other antigenic determinant, epitope, part or domain on HER2; and preferably comprise single variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic and are at least directed against the Herceptin® binding site on HER2 as well as against the Omnitarg binding site on HER2. The amino acid sequences and polypeptides of the invention may be directed against domain IV of HER2. The amino acid sequences and polypeptides of the invention may be directed against domain II of HER2. The amino acid sequences and polypeptides of the invention may be directed against domain IV of HER2 as well as against domain II of HER2. In a preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the C-terminus of domain IV of HER2. In another preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the C-terminus of domain IV of HER2 as well as against domain H of HER2. In another preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the center of domain II of HER2. In another preferred aspect, the amino acid sequences and polypeptides of the invention are directed against domain IV of HER2 as well as against the center of domain II of HER2. In another preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the C-terminus of domain IV of HER2 as well as against the center of domain II of HER2.

Again, the above biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the Omnitarg binding site and the Herceptin®-binding site; and preferably comprise single variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic with both paratopes directed against the Herceptin® binding site on HER2. The amino acid sequences and polypeptides of the invention may be directed against domain IV of HER2 (one paratope or both paratopes). In a preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the C-terminus of domain IV of HER2 (one paratope or both paratopes).

In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic with both paratopes directed against the Omnitarg binding site on HER2. The amino acid sequences and polypeptides of the invention may be directed against domain. II of HER2 (one paratope or both paratopes). In a preferred aspect, the amino acid sequences and polypeptides of the invention are directed against the center of domain. II of HER2 (one paratope or both paratopes).

Again, the above biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind both binding sites; and preferably comprise single variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are at least capable, upon binding to HER-2, (A) to modulate (as defined herein) HER-2 or HER-2 mediated signalling by inhibiting ligand activation of an ErbB hetero-oligomer comprising HER2 and HER3, HER4 or EGFR, or more generally capable of modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Omnitarg; and (B) to (i) recruit immune effector cells such as macrophages and monocytes to the tumor (for this purpose, most preferably a polypeptide of the invention is used that contains an Fc portion that confers upon the polypeptide the ability to recruit immune effector cells such as macrophages and monocytes to the tumor); and/or (ii) modulate (as defined herein) HER-2 or HER-2 mediated signalling by downregulating HER2 levels and/or by down-regulating HER2-mediated signaling pathways; and/or (iii) modulate (as defined herein) HER-2 or HER-2 mediated signalling by blocking or inhibiting metalloproteinase-mediated HER2 ectodomain shedding; or more generally by modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Herceptin®.

For example, for this purpose, such a biparatopic (or multiparatopic) polypeptide of the invention may comprise

-   -   at least one first amino acid sequence of the invention that is         capable, upon binding to HER-2, to modulate (as defined herein)         HER-2 or HER-2 mediated signalling by inhibiting ligand         activation of an ErbB hetero-oligomer comprising HER2 and HER3,         HER4 or EGFR, or more generally capable of modulating (as         defined herein) HER-2 or HER-2 mediated signalling via the same         mechanism of action as Omnitarg. Such an amino acid sequence of         the invention is preferably an amino acid sequence that is         directed against the Omnitarg binding site on HER2 (and in         particular against domain II of HER2, and more in particular         against the middle of domain II of HER2) and/or capable of         competing with Omnitarg for binding to HER-2;         and further comprise either     -   an Fc portion that confers upon the polypeptide the ability to         recruit immune effector cells such as macrophages and monocytes         to the tumor),         and/or     -   at least one amino acid sequence of the invention that is         capable, upon binding to HER-2, to (1) modulate (as defined         herein) HER-2 or HER-2 mediated signalling by downregulating         HER2 levels and/or by downregulating HER2-mediated signaling         pathways; and/or (2) modulate (as defined herein) HER-2 or HER-2         mediated signalling by blocking or inhibiting         metalloproteinase-mediated HER2 ectodomain shedding; or more         generally (3) modulate (as defined herein) HER-2 or HER-2         mediated signalling via the same mechanism of action as         Herceptin®. Such an amino acid sequence of the invention is         preferably an amino acid sequence that is directed against the         Herceptin® binding site on HER2 (and in particular against         domain IV of HER2, and more in particular against the C-terminus         of domain IV of HER2) and/or capable of competing with         Herceptin® for binding to HER-2.

Again, such a biparatopic (or multiparatopic) polypeptide of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind at least two different antigenic determinants, epitopes, parts or domains or HER-2, such as the Omnitarg binding site and the Herceptin®-binding site; and preferably comprise single variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are at least capable, upon binding to HER-2, (A) to modulate (as defined herein) HER-2 or HER-2 mediated signalling by inhibiting ligand activation of an ErbB hetero-oligomer comprising HER2 and HER3, HER4 or EGFR, or more generally capable of modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Omnitarg; and (B) to bind to the Herceptin® binding site on HER2 (and in particular to domain IV of HER2, and more in particular to the C-terminus of domain IV of HER2) and/or to compete with Herceptin® for binding to HER-2.

For example, for this purpose, such a biparatopic (or multiparatopic) polypeptide of the invention may comprise

-   -   at least one first amino acid sequence of the invention that is         capable, upon binding to HER-2, to modulate (as defined herein)         HER-2 or HER-2 mediated signalling by inhibiting ligand         activation of an ErbB hetero-oligomer comprising HER2 and HER3,         HER4 or EGFR, or more generally capable of modulating (as         defined herein) HER-2 or HER-2 mediated signalling via the same         mechanism of action as Omnitarg;         and further comprise either     -   at least one amino acid sequence of the invention that is         directed against the Herceptin® binding site on HER2 (and in         particular against domain IV of HER2, and more in particular         against the C-terminus of domain IV of HER2) and/or capable of         competing with Herceptin® for binding to HER-2.

Again, such a biparatopic (or multiparatopic) polypeptide of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind at least two different antigenic determinants, epitopes, parts or domains or HER-2, at least including the Herceptin®-binding site; and preferably comprise single variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are at least capable, upon binding to HER-2, (A) to bind to the Omnitarg binding site on HER2 (and in particular to domain II of HER2, and more in particular to the middle of domain II of HER2) and/or capable of competing with Omnitarg for binding to HER-2; and (B) to (i) recruit immune effector cells such as macrophages and monocytes to the tumor (for this purpose, most preferably a polypeptide of the invention is used that contains an Fc portion that confers upon the polypeptide the ability to recruit immune effector cells such as macrophages and monocytes to the tumor); and/or (ii) modulate (as defined herein) HER-2 or HER-2 mediated signalling by downregulating HER2 levels and/or by downregulating HER2-mediated signaling pathways; and/or (iii) modulate (as defined herein) HER-2 or HER-2 mediated signalling by blocking or inhibiting metalloproteinase-mediated HER2 ectodomain shedding; or more generally by modulating (as defined herein) HER-2 or HER-2 mediated signalling via the same mechanism of action as Herceptin®.

For example, for this purpose, such a biparatopic (or multiparatopic) polypeptide of the invention may comprise:

-   -   at least one first amino acid sequence of the invention that is         directed against the Omnitarg binding site on HER2 (and in         particular against domain II of HER2, and more in particular         against the middle of domain II of HER2) and/or capable of         competing with Omnitarg for binding to HER-2;         and further comprise either     -   an Fc portion that confers upon the polypeptide the ability to         recruit immune effector cells such as macrophages and monocytes         to the tumor),         and/or     -   at least one amino acid sequence of the invention that is         capable, upon binding to HER-2, to (1) modulate (as defined         herein) HER-2 or HER-2 mediated signalling by downregulating         HER2 levels and/or by downregulating HER2-mediated signaling         pathways; and/or (2) modulate (as defined herein) HER-2 or HER-2         mediated signalling by blocking or inhibiting         metalloproteinase-mediated HER2 ectodomain shedding; or more         generally (3) modulate (as defined herein) HER-2 or HER-2         mediated signalling via the same mechanism of action as         Herceptin®. Such an amino acid sequence of the invention is         preferably an amino acid sequence that is directed against the         Herceptin® binding site on HER2 (and in particular against         domain IV of HER2, and more in particular against the C-terminus         of domain IV of HER2) and/or capable of competing with         Herceptin® for binding to HER-2.

Again, such a biparatopic (or multiparatopic) polypeptide of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind at least two different antigenic determinants, epitopes, parts or domains or HER-2, at least including the Omnitarg binding site; and preferably comprise single variable domains and more preferably Nanobodies).

It is also expected that the amino acid sequences and polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of HER2; or at least to those analogs, variants, mutants, alleles, parts and fragments of HER2 that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the amino acid sequences and polypeptides of the invention bind in HER2 (e.g. in wild-type HER2). Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and/or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to (wild-type) HER2. It is also included within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to some analogs, variants, mutants, alleles, parts and fragments of HER2, but not to others.

When HER2 exists in a monomeric form and in one or more multimeric forms, it is within the scope of the invention that the amino acid sequences and polypeptides of the invention only bind to HER2 in monomeric form, only bind to HER2 in multimeric form, or bind to both the monomeric and the multimeric form. Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to the monomeric form with an affinity and/or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to the multimeric form.

In a non-limiting aspect, the amino acid sequences and polypeptides of the invention only bind to HER2 in monomeric form while not binding to HER2 in dimerized state. In another non-limiting aspect, the amino acid sequences and polypeptides of the invention only bind to HER2 in dimerized state while not binding to HER2 in monomeric form. In another non-limiting aspect, the amino acid sequences and polypeptides of the invention bind to HER2 in monomeric form as well as to HER2 in dimerized state.

Also, when HER2 can associate with other proteins or polypeptides (e.g. with other ERBB receptors, also referred to as heterodimerization) to form protein complexes (e.g. with multiple subunits), it is within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to HER2 in its non-associated state, bind HER2 in its associated state, or bind to both. In a non-limiting aspect, the amino acid sequences and polypeptides of the invention only bind to HER2 when HER-2 is in its monomeric form while not binding to HER2 when HER-2 is in its dimerized state. In another non-limiting aspect, the amino acid sequences and polypeptides of the invention only bind to HER2 when HER-2 is in its dimerized state while not binding to HER2 when HER-2 is in monomeric form. In another non-limiting aspect, the amino acid sequences and polypeptides of the invention bind to HER2 in monomeric form as well as to HER2 in dimerized state. In all these cases, the amino acid sequences and polypeptides of the invention may bind to such multimers or associated protein complexes with an affinity and/or specificity that may be the same as or different from (i.e. higher than or lower than) the affinity and/or specificity with which the amino acid sequences and polypeptides of the invention bind to HER2 in its monomeric and non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptides that contain two or more amino acid sequences directed against HER2 may bind with higher avidity to HER2 than the corresponding monomeric amino acid sequence(s). For example, and without limitation, proteins or polypeptides that contain two or more amino acid sequences directed against different epitopes of HER2 may (and usually will) bind with higher avidity than each of the different monomers, and proteins or polypeptides that contain two or more amino acid sequences directed against HER2 may (and usually will) bind also with higher avidity to a multimer of HER2.

Generally, amino acid sequences and polypeptides of the invention will at least bind to those forms of HER2 (including monomeric, multimeric and associated forms) that are the most relevant from a biological and/or therapeutic point of view, as will be clear to the skilled person.

It is also within the scope of the invention to use parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the amino acid sequences and polypeptides of the invention, and/or to use proteins or polypeptides comprising or essentially consisting of one or more of such parts, fragments, analogs, mutants, variants, alleles and/or derivatives, as long as these are suitable for the uses envisaged herein. Such parts, fragments, analogs, mutants, variants, alleles and/or derivatives will usually contain (at least part of) a functional antigen-binding site for binding against HER2; and more preferably will be capable of specific binding to HER2, and even more preferably capable of binding to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein. Some non-limiting examples of such parts, fragments, analogs, mutants, variants, alleles, derivatives, proteins and/or polypeptides will become clear from the further description herein. Additional fragments or polypeptides of the invention may also be provided by suitably combining (i.e. by linking or genetic fusion) one or more (smaller) parts or fragments as described herein.

In one specific, but non-limiting aspect of the invention, which will be further described herein, such analogs, mutants, variants, alleles, derivatives have an increased half-life in serum (as further described herein) compared to the amino acid sequence from which they have been derived. For example, an amino acid sequence of the invention may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide a derivative of an amino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that comprises an immunoglobulin fold or may be an amino acid sequence that, under suitable conditions (such as physiological conditions) is capable of forming an immunoglobulin fold (i.e. by folding). Reference is inter alia made to the review by Halaby et al., J. (1999) Protein Eng. 12, 563-71. Preferably, when properly folded so as to form an immunoglobulin fold, such an amino acid sequence is capable of specific binding (as defined herein) to HER2; and more preferably capable of binding to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein. Also, parts, fragments, analogs, mutants, variants, alleles and/or derivatives of such amino acid sequences are preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold.

In particular, but without limitation, the amino acid sequences of the invention may be amino acid sequences that essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR's, as further described herein).

The amino acid sequences of the invention may in particular be an immunoglobulin sequence or a suitable fragment thereof, and more in particular be an immunoglobulin variable domain sequence or a suitable fragment thereof, such as light chain variable domain sequence (e.g. a V_(L)-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a V_(H)-sequence) or a suitable fragment thereof. When the amino acid sequence of the invention is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a V_(H) sequence that is derived from a human antibody) or be a so-called. V_(HH)-sequence (as defined herein) that is derived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to the origin of the amino acid sequence of the invention (or of the nucleotide sequence of the invention used to express it), nor as to the way that the amino acid sequence or nucleotide sequence of the invention is (or has been) generated or obtained. Thus, the amino acid sequences of the invention may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences. In a specific but non-limiting aspect of the invention, the amino acid sequence is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to “humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized V_(HH) sequences or Nanobodies), “camelized” (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing. Reference is for example made to the standard handbooks, as well as to the further description and prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a “dAb” (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® (as defined herein, and including but not limited to a V_(HH) sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term “dAb's”, reference is for example made to Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; as well as to for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called “IgNAR domains”, see for example WO 05/18629).

In particular, the amino acid sequence of the invention may be a Nanobody® (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V.] Such Nanobodies directed against HER2 will also be referred to herein as “Nanobodies of the invention”.

For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described Nanobodies of the so-called “V_(H)3 class” (i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29), which Nanobodies form a preferred aspect of this invention. It should however be noted that the invention in its broadest sense generally covers any type of Nanobody directed against HER2, and for example also covers the Nanobodies belonging to the so-called “V_(H)4 class” (i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V_(H)4 class such as DP-78), as for example described in WO 07/118,670.

Generally, Nanobodies (in particular V_(HH) sequences and partially humanized Nanobodies) can in particular be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which one or more of the Hallmark residues are as further         defined herein.

In particular, a Nanobody can be an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which the framework sequences are as further defined herein.

More in particular, a Nanobody can be an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   i) preferably one or more of the amino acid residues at positions     11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat     numbering are chosen from the Hallmark residues mentioned in Table     A-3 below;     and in which:

-   ii) said amino acid sequence has at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NOs: 1 to     22, in which for the purposes of determining the degree of amino     acid identity, the amino acid residues that form the CDR sequences     (indicated with X in the sequences of SEQ ID NOs: 1 to 22) are     disregarded.

In these Nanobodies, the CDR sequences are generally as further defined herein.

Thus, the invention also relates to such Nanobodies that can bind to (as defined herein) and/or are directed against HER2, to suitable fragments thereof, as well as to polypeptides that comprise or essentially consist of one or more of such Nanobodies and/or suitable fragments.

SEQ ID NOs: 2051-2325 give the amino acid sequences of a number of V_(HH) sequences that have been raised against HER2.

In particular, the invention in some specific aspects provides:

-   -   amino acid sequences that are directed against (as defined         herein) HER2 and that have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 2051-2325. These         amino acid sequences may further be such that they are directed         against an interaction site (as defined herein) on HER2 (such as         the Herceptin® or Omnitarg binding site);     -   amino acid sequences that cross-block (as defined herein) the         binding of at least one of the amino acid sequences of SEQ ID         NO's: 2051-2325 to HER2 and/or that compete with at least one of         the amino acid sequences of SEQ ID NO's: 2051-2325 for binding         to HER2. Again, these amino acid sequences may further be such         that they are directed against an interaction site (as defined         herein) on HER2 (such as Herceptin® or Omnitarg binding site);

which amino acid sequences may be as further described herein (and may for example be Nanobodies); as well as polypeptides of the invention that comprise one or more of such amino acid sequences (which may be as further described herein, and may for example be bispecific and/or biparatopic polypeptides as described herein), and nucleic acid sequences that encode such amino acid sequences and polypeptides. Such amino acid sequences and polypeptides do not include any naturally occurring ligands.

Accordingly, some particularly preferred Nanobodies of the invention are Nanobodies which can bind (as further defined herein) to and/or are directed against to HER2 and which:

-   i) have at least 80% amino acid identity with at least one of the     amino acid sequences of SEQ ID NOs: 2051-2325, in which for the     purposes of determining the degree of amino acid identity, the amino     acid residues that form the CDR sequences are disregarded. In this     respect, reference is also made to Table A-1, which lists the     framework 1 sequences (SEQ ID NOs: 126-400), framework 2 sequences     (SEQ ID NOs: 676-950), framework 3 sequences (SEQ ID NOs: 1226-1500)     and framework 4 sequences (SEQ ID NOs: 1776-2050) of the Nanobodies     of SEQ ID NOs: 2051-2325 (with respect to the amino acid residues at     positions 1 to 4 and 27 to 30 of the framework 1 sequences,     reference is also made to the comments made below. Thus, for     determining the degree of amino acid identity, these residues are     preferably disregarded);     and in which: -   ii) preferably one or more of the amino acid residues at positions     11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat     numbering are chosen from the Hallmark residues mentioned in Table     A-3 below.

In these Nanobodies, the CDR sequences are generally as further defined herein.

Again, such Nanobodies may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring V_(HH) sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to “humanized” (as defined herein) Nanobodies, “camelized” (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as Nanobodies that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. Also, when a Nanobody comprises a V_(HH) sequence, said Nanobody may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized Nanobodies of the invention. Similarly, when a Nanobody comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence), said Nanobody may optionally be further suitably humanized, again as described herein, again so as to provide one or more further (partially or fully) humanized Nanobodies of the invention.

In particular, humanized Nanobodies may be amino acid sequences that are as generally defined for Nanobodies in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanizing substitution (as defined herein). Some preferred, but non-limiting humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V_(HH) sequence with the corresponding framework sequence of one or more closely related human V_(H) sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said sequence (in any manner known per se, as further described herein) and the resulting humanized V_(HH) sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a Nanobody may be partially humanized or fully humanized.

Some particularly preferred humanized Nanobodies of the invention are humanized variants of the Nanobodies of SEQ ID NOs: 2051-2325.

Thus, some other preferred Nanobodies of the invention are Nanobodies which can bind (as further defined herein) to HER2 and which:

-   i) are a humanized variant of one of the amino acid sequences of SEQ     ID NOs: 2051-2325; and/or -   ii) have at least 80% amino acid identity with at least one of the     amino acid sequences of SEQ ID NOs: 2051-2325, in which for the     purposes of determining the degree of amino acid identity, the amino     acid residues that form the CDR sequences are disregarded;     and in which: -   i) preferably one or more of the amino acid residues at positions     11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat     numbering are chosen from the Hallmark residues mentioned in Table     A-3 below.

According to another specific aspect of the invention, the invention provides a number of stretches of amino acid residues (i.e. small peptides) that are particularly suited for binding to HER2. These stretches of amino acid residues may be present in, and/or may be corporated into, an amino acid sequence of the invention, in particular in such a way that they form (part of) the antigen binding site of an amino acid sequence of the invention. As these stretches of amino acid residues were first generated as CDR sequences of heavy chain antibodies or V_(HH) sequences that were raised against HER2 (or may be based on and/or derived from such CDR sequences, as further described herein), they will also generally be referred to herein as “CDR sequences” (i.e. as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively). It should however be noted that the invention in its broadest sense is not limited to a specific structural role or function that these stretches of amino acid residues may have in an amino acid sequence of the invention, as long as these stretches of amino acid residues allow the amino acid sequence of the invention to bind to HER2. Thus, generally, the invention in its broadest sense comprises any amino acid sequence that is capable of binding to HER2 and that comprises one or more CDR sequences as described herein, and in particular a suitable combination of two or more such CDR sequences, that are suitably linked to each other via one or more further amino acid sequences, such that the entire amino acid sequence forms a binding domain and/or binding unit that is capable of binding to HER2. It should however also be noted that the presence of only one such CDR sequence in an amino acid sequence of the invention may by itself already be sufficient to provide an amino acid sequence of the invention that is capable of binding to HER2; reference is for example again made to the so-called “Expedite fragments” described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that comprises at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof). In particular, an amino acid sequence of the invention may be an amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof).

Generally, in this aspect of the invention, the amino acid sequence of the invention may be any amino acid sequence that comprises at least one stretch of amino acid residues, in which said stretch of amino acid residues has an amino acid sequence that corresponds to the sequence of at least one of the CDR sequences described herein. Such an amino acid sequence may or may not comprise an immunoglobulin fold. For example, and without limitation, such an amino acid sequence may be a suitable fragment of an immunoglobulin sequence that comprises at least one such CDR sequence, but that is not large enough to form a (complete) immunoglobulin fold (reference is for example again made to the “Expedite fragments” described in WO 03/050531). Alternatively, such an amino acid sequence may be a suitable “protein scaffold” that comprises least one stretch of amino acid residues that corresponds to such a CDR sequence (i.e. as part of its antigen binding site). Suitable scaffolds for presenting amino acid sequences will be clear to the skilled person, and for example comprise, without limitation, to binding scaffolds based on or derived from immunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived from protein A domains (such as Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32).

Again, any amino acid sequence of the invention that comprises one or more of these CDR sequences is preferably such that it can specifically bind (as defined herein) to HER2, and more in particular such that it can bind to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect of the invention may be any amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least two amino acid sequences that are chosen from the group consisting of the CDR1 sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that (i) when the first amino acid sequence is chosen from the CDR1 sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein or the CDR3 sequences described herein; (ii) when the first amino acid sequence is chosen from the CDR2 sequences described herein, the second amino acid sequence is chosen from the CDR1 sequences described herein or the CDR3 sequences described herein; or (iii) when the first amino acid sequence is chosen from the CDR3 sequences described herein, the second amino acid sequence is chosen from the CDR 1 sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention may be amino acid sequences that comprise at least one antigen binding site, wherein said antigen binding site comprises at least three amino acid sequences that are chosen from the group consisting of the CDR1 sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that the first amino acid sequence is chosen from the CDR1 sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein, and the third amino acid sequence is chosen from the CDR3 sequences described herein. Preferred combinations of CDR1, CDR2 and CDR3 sequences will become clear from the further description herein. As will be clear to the skilled person, such an amino acid sequence is preferably an immunoglobulin sequence (as further described herein), but it may for example also be any other amino acid sequence that comprises a suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against HER2, that comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     401-675; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     401-675; -   d) the amino acid sequences of SEQ ID NO's: 951-1225; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225; -   g) the amino acid sequences of SEQ ID NO's: 1501-1775; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775;     or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence     according to b) and/or c) is preferably, and compared to the     corresponding amino acid sequence according to a), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to b) and/or c) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to a);     and/or -   iii) the amino acid sequence according to b) and/or c) may be an     amino acid sequence that is derived from an amino acid sequence     according to a) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence     according to e) and/or f) is preferably, and compared to the     corresponding amino acid sequence according to d), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to e) and/or f) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to d);     and/or -   iii) the amino acid sequence according to e) and/or f) may be an     amino acid sequence that is derived from an amino acid sequence     according to d) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence     according to h) and/or i) is preferably, and compared to the     corresponding amino acid sequence according to g), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to h) and/or i) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to g);     and/or -   iii) the amino acid sequence according to h) and/or i) may be an     amino acid sequence that is derived from an amino acid sequence     according to g) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   i) the amino acid sequences of SEQ ID NO's: 401-675; -   ii) the amino acid sequences of SEQ ID NO's: 951-1225; and -   iii) the amino acid sequences of SEQ ID NO's: 1501-1775;     or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against HER2.

In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against HER2, that comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     401-675; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     401-675; -   d) the amino acid sequences of SEQ ID NO's: 951-1225; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225; -   g) the amino acid sequences of SEQ ID NO's: 1501-1775; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775;     such that (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences according to a), b)     or c), the second stretch of amino acid residues corresponds to one     of the amino acid sequences according to d), e), g), h) or i); (ii)     when the first stretch of amino acid residues corresponds to one of     the amino acid sequences according to d), e) or f), the second     stretch of amino acid residues corresponds to one of the amino acid     sequences according to a), b), c), g), h) or i); or (iii) when the     first stretch of amino acid residues corresponds to one of the amino     acid sequences according to g), h) or i), the second stretch of     amino acid residues corresponds to one of the amino acid sequences     according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   i) the amino acid sequences of SEQ ID NO's: 401-675; -   ii) the amino acid sequences of SEQ ID NO's: 951-1225; and -   iii) the amino acid sequences of SEQ ID NO's: 1501-1775;     such that, (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's:     401-675, the second stretch of amino acid residues corresponds to     one of the amino acid sequences of SEQ ID NO's: 951-1225 or of SEQ     ID NO's: 1501-1775; (ii) when the first stretch of amino acid     residues corresponds to one of the amino acid sequences of SEQ ID     NO's: 951-1225, the second stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's:     401-675 or of SEQ ID NO's: 1501-1775; or (iii) when the first     stretch of amino acid residues corresponds to one of the amino acid     sequences of SEQ ID NO's: 1501-1775, the second stretch of amino     acid residues corresponds to one of the amino acid sequences of SEQ     ID NO's: 401-675 or of SEQ ID NO's: 951-1225.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against HER2.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against HER2, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     401-675; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with, at least one of the amino acid sequences of SEQ ID NO's:     401-675;     the second stretch of amino acid residues is chosen from the group     consisting of: -   d) the amino acid sequences of SEQ ID NO's: 951-1225; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;     and the third stretch of amino acid residues is chosen from the     group consisting of: -   g) the amino acid sequences of SEQ ID NO's: 1501-1775; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775.

Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 401-675; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 951-1225; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 1501-1775.

Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against HER2. Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 2051-2325. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 2051-2325, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to HER2; and more in particular bind to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     401-675;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     401-675;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 951-1225;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775.

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 401-675; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 951-1225; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 1501-1775.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     401-675;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     401-675;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 951-1225;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775; or any suitable fragment of such an amino acid sequence.

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 401-675 and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 951-1225; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 1501-1775.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to HER2; and more in particular bind to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 2051-2325. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 2051-2325, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.

In such an amino acid sequence of the invention, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

The framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. a V_(H)-sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a V_(HH)-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional V_(H) sequences that have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequence of the invention is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); is a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody); is a “dAb” (or an amino acid sequence that is suitable for use as a dAb); or is a Nanobody® (including but not limited to V_(HH) sequence). Again, suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acid sequences of the invention may contain one or more of Hallmark residues (as defined herein), such that the amino acid sequence of the invention is a Nanobody®. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

Again, as generally described herein for the amino acid sequences of the invention, it is also possible to use suitable fragments (or combinations of fragments) of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (for example, in the same order as these CDR's and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived). Such fragments may also again be such that they comprise or can form an immunoglobulin fold, or alternatively be such that they do not comprise or cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequence as described herein (and in particular a CDR3 sequence), that is flanked on each side by (part of) a framework sequence (and in particular, part of the framework sequence(s) that, in the immunoglobulin sequence from which the fragment is derived, are adjacent to said CDR sequence. For example, a CDR3 sequence may be preceded by (part of) a FR3 sequence and followed by (part of) a FR4 sequence). Such a fragment may also contain a disulphide bridge, and in particular a disulphide bridge that links the two framework regions that precede and follow the CDR sequence, respectively (for the purpose of forming such a disulphide bridge, cysteine residues that naturally occur in said framework regions may be used, or alternatively cysteine residues may be synthetically added to or introduced into said framework regions). For a further description of these “Expedite fragments”, reference is again made to WO 03/050531, as well as to WO 08/068,280 of Ablynx N.V.

In another aspect, the invention relates to a compound or construct, and in particular a protein or polypeptide (also referred to herein as a “compound of the invention” or “polypeptide of the invention”, respectively) that comprises or essentially consists of one or more amino acid sequences of the invention (or suitable fragments thereof), and optionally further comprises one or more other groups, residues, moieties or binding units. As will become clear to the skilled person from the further disclosure herein, such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the amino acid sequence of the invention (and/or to the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence of the invention.

For example, such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, “dAb”'s, amino acid sequences that are suitable for use as a dAb, or Nanobodies.

Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more amino acid sequences of the invention so as to provide a “derivative” of an amino acid sequence or polypeptide of the invention, as further described herein.

Also within the scope of the present invention are compounds or constructs, that comprises or essentially consists of one or more derivatives as described herein, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more amino acid sequences of the invention and the one or more groups, residues, moieties or binding units may be linked directly to each other and/or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be amino acid sequences, so that the resulting compound or construct is a fusion (protein) or fusion (polypeptide).

The compounds or polypeptides of the invention can generally be prepared by a method which comprises at least one step of suitably linking the one or more amino acid sequences of the invention to the one or more further groups, residues, moieties or binding units, optionally via the one or more suitable linkers, so as to provide the compound or polypeptide of the invention. Polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.

The process of designing/selecting and/or preparing a compound or polypeptide of the invention, starting from an amino acid sequence of the invention, is also referred to herein as “formatting” said amino acid sequence of the invention; and an amino acid of the invention that is made part of a compound or polypeptide of the invention is said to be “formatted” or to be “in the format of” said compound or polypeptide of the invention. Examples of ways in which an amino acid sequence of the invention can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted amino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention or a polypeptide of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin; see for example EP 0 368 684 B1, page 4); or polypeptides of the invention that comprise at least one amino acid sequence of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the amino acid sequence of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, “dAb”'s, amino acid sequences that are suitable for use as a dAb, or Nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrine; reference is made to the further description and references mentioned herein); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more small, proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to WO 08/068,280 of Ablynx N.V.

Generally, the compounds or polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the compounds or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention have a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodes an amino acid sequence of the invention or a polypeptide of the invention (or a suitable fragment thereat).

Such a nucleic acid will also be referred to herein as a “nucleic acid of the invention” and may for example be in the form of a genetic construct, as further described herein.

In another aspect, the invention relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) an amino acid sequence of the invention and/or a polypeptide of the invention; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

The invention further relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention (or a suitable fragment thereof) and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

The invention also relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same, in (methods or compositions for) modulating HER2, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a cancer and/or tumor); and/or in methods for killing a tumor cell or inhibiting or preventing proliferation of a tumour cell (either in vitro or in vivo) by suitably contacting said tumor cell with an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same. In a preferred, but non-limiting aspect, a biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

The invention also relates to methods for modulating HER2, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a cancer and/or tumor), which method comprises at least the step of contacting HER2 with at least one amino acid sequence, Nanobody or polypeptide of the invention, or with a composition comprising the same, in a manner and in an amount suitable to modulate HER2, with at least one amino acid sequence, Nanobody or polypeptide of the invention. In a preferred, but non-limiting aspect, a biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

The invention also relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating HER2, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a cancer and/or tumor). In a preferred, but non-limiting aspect, a biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In the context of the present invention, “modulating” or “to modulate” generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, HER2, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein). In particular, “modulating” or “to modulate” may mean either reducing or inhibiting the activity of, or alternatively increasing the activity of HER2, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of HER2 in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody or polypeptide of the invention.

As will be clear to the skilled person, “modulating” may also involve effecting a change (which may either be an increase or a decrease) in the sensitivity of HER2 for one or more conditions in the medium or surroundings in which HER2 is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the amino acid sequence, Nanobody or polypeptide of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, such as the assays described herein or in the prior art cited herein.

“Modulating” may also mean effecting a change (i.e. an activity as an agonist or as an antagonist, respectively) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which HER2 (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, such as the assays described herein or in the prior art cited herein. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody or polypeptide of the invention.

Modulating may also involve activating HER2 or the mechanism or pathway in which it is involved. Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner. Modulating may for example also involve reducing or inhibiting the binding of HER2 to another ERBB receptor (also referred to as heterodimerization) and/or competing with another ERBB receptor for binding to HER2.

Without being limiting, in one aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block binding of Herceptin® to HER2. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit binding of Herceptin® to HER2 by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to binding of Herceptin® to HER2 in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same.

In another aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block binding of Omnitarg to HER2. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit binding of Omnitarg to HER2 by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to binding of Omnitarg to HER2 in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same.

In another aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block binding of Herceptin® and Omnitarg to HER, preferably essentially simultaneously. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit binding of Herceptin® to HER2 by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to binding of Herceptin® to HER2 in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same; and the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit binding of Omnitarg to HER2 by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to binding of Omnitarg to HER2 in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an antagonist of HER2 and/or of the signalling that is mediated by HER-2 and/or by the ligand(s) of HER-2 (i.e. of the signalling that is caused by binding of growth factors of the EGF family to HER-2) and will inhibit and/or block such signalling (i.e. by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the signalling without the presence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same, as determined in a suitable assay); and/or will inhibit or block tumor (e.g. SKBR3) cell proliferation. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit tumor (e.g. SKBR3) cell proliferation by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the tumor (e.g. SKBR3) cell proliferation in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor (e.g. SKBR3) cell proliferation equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor (e.g. SKBR3) cell proliferation equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor (e.g. SKBR3) cell proliferation equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of diseases and disorders that can be prevented or treated by increasing HER-2 signalling in one or more cells or tissues of a patient to be treated, such as certain cardiac disorders (i.e. those characterised by reduced HER-2-mediated signalling or those that are a side-effect from treating a patient with a HER-2 antagonist), the amino acid sequence. Nanobody or polypeptide of the invention or the composition comprising the same is an agonist of HER2 and will induce cell proliferation. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably increase the signalling that is mediated by HER-2 and/or by the ligand(s) of HER-2 (i.e. by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the signalling without the presence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same, as determined in a suitable assay); and/or will induce cell proliferation by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the cell proliferation in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred, but non-limiting aspect, a suitable agonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an antagonist of HER2 and will inhibit, downregulate and/or block cell signalling. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit and/or downregulate cell signalling by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the cell signalling in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit, downregulate and/or block cell signalling equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit, downregulate and/or block cell signalling equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit, downregulate and/or block cell signalling equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of diseases and disorders that can be prevented or treated by increasing HER-2 signalling in one or more cells or tissues of a patient to be treated, such as certain cardiac disorders (i.e. those characterised by reduced HER-2-mediated signalling or those that are a side-effect from treating a patient with a HER-2 antagonist), the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an agonist of HER2 and will induce cell signalling. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably induce cell signalling by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the cell signalling in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred, but non-limiting aspect, a suitable agonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an antagonist of HER2 and will inhibit and/or block tumor (e.g. SKBR3) cell proliferation in vivo. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit tumor (e.g. SKBR3) cell proliferation in vivo by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the tumor (e.g. SKBR3) cell proliferation in vivo in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor (e.g. SKBR3) cell proliferation in vivo equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor (e.g. SKBR3) cell proliferation in vivo equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor (e.g. SKBR3) cell proliferation in vivo equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an antagonist of HER2 and will inhibit, downregulate and/or block ligand-mediated ErbB signalling. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit and/or downregulate ligand-mediated ErbB signalling by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the ligand-mediated ErbB signalling in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block ligand-mediated ErbB signalling equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block ligand-mediated ErbB signalling equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block ligand-mediated ErbB signalling equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an antagonist of HER2 and will inhibit and/or block HER2 ectodomain cleavage. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit HER2 ectodomain cleavage by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the HER2 ectodomain cleavage in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block HER2 ectodomain cleavage equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block HER2 ectodomain cleavage equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block HER2 ectodomain cleavage equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an antagonist of HER2 and will inhibit and/or block Heregulin-mediated activation of MAPK/Erk1/2. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit Heregulin-mediated activation of MAPK/Erk1/2 by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the Heregulin-mediated activation of MAPK/Erk1/2 in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block Heregulin-mediated activation of MAPK/Erk1/2 equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block Heregulin-mediated activation of MAPK/Erk1/2 equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block Heregulin-mediated activation of MAPK/Erk1/2 equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an antagonist of HER2 and will inhibit and/or block PI3K/Akt signalling. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit PI3K/Akt signalling by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the PI3K/Akt signalling in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block PI3K/Akt signalling equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block. PI3K/Akt signalling equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block PI3K/Akt signalling equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same is an antagonist of HER2 and will inhibit, downregulate and/or block cell signalling in vivo. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit and/or downregulate cell signalling in vivo by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the cell signalling in vivo in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block cell signalling in vivo equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block cell signalling in vivo equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block cell signalling in vivo equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will induce apoptosis in tumor cells. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably induce apoptosis in tumor cells by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to apoptosis in tumor cells in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will induce apoptosis in tumor cells equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will induce apoptosis in tumor cells equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will induce apoptosis in tumor cells equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block heterodimerization between ERBB receptors. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit and/or block heterodimerization between ERBB receptors by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the heterodimerization between ERBB receptors in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence. Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block heterodimerization between ERBB receptors equally or better than Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor vascularisation. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit tumor vascularisation by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the tumor vascularisation in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor vascularisation equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor vascularisation equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block tumor vascularisation equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block TNF induced signalling and/or cell proliferation. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit TNF induced signalling and/or cell proliferation by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the TNF induced signalling and/or cell proliferation in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block TNF induced signalling and/or cell proliferation equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block TNF induced signalling and/or cell proliferation equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block TNF induced signalling and/or cell proliferation equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will downregulate HER2 levels. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably will downregulate HER2 levels by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the HER2 levels in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will down-regulate HER2 levels equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will downregulate HER2 levels equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will downregulate HER2 levels equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in the prevention and treatment of tumors and cancer, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block metalloproteinase-mediated HER2 ectodomain shedding. The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will preferably inhibit metalloproteinase-mediated HER2 ectodomain shedding by at least 1%, preferably at least 5%, such as at least 10%, for example 25% or more or even 50% or more and up to 75% or even more than 90% or more, compared to the metalloproteinase-mediated HER2 ectodomain shedding in the absence of the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same. In a preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block metalloproteinase-mediated HER2 ectodomain shedding equally or better than Herceptin®. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block metalloproteinase-mediated HER2 ectodomain shedding equally or better than Omnitarg. In another preferred aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same will inhibit and/or block metalloproteinase-mediated HER2 ectodomain shedding equally or better than Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, a suitable antagonistic biparatopic (or multiparatopic) polypeptide of the invention is used, and more preferably one of the preferred biparatopic (or multiparatopic) polypeptides of the invention, as further described herein.

The amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same should at least “modulate” or effect a change (i.e. an activity as an agonist or as an antagonist, respectively) with respect to at least one biological or physiological mechanisms, effects, responses, functions, pathways or activities (also referred to herein as “having at least one mode of action”) in which HER2 (or in which its pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. In one aspect, the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same may “modulate” or effect a change with respect to more than one (such as two, three, four or even more) biological or physiological mechanisms, effects, responses, functions, pathways or activities (i.e. the amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same may have more than one mode of action). In this respect, the present inventors surprisingly found that the biparatopic amino acid sequence, Nanobody or polypeptide of the invention or the composition comprising the same could combine two different modes of action (such as e.g. they could inhibit and/or block two different cell signalling pathways; they could e.g. inhibit and/or block heterodimerization between ERBB receptors and at the same time downregulate HER2 levels).

The different modes of action are mediated each by one of the binding units (as further defined herein) of the biparatopic amino acid sequence, Nanobody or polypeptide of the invention, wherein each binding unit binds at a different binding site of HER2. In a preferred aspect, the biparatopic amino acid sequence, Nanobody or polypeptide of the invention combine the modes of action of Herceptin® and Omnitarg.

Accordingly, the present invention also relates to a biparatopic amino acid sequence, Nanobody or polypeptide of the invention or a composition comprising the same that combines two different modes of action each mediated by one of the binding units of the biparatopic amino acid sequence, Nanobody or polypeptide of the invention, wherein each binding unit binds at a different binding site of HER2.

Accordingly, the present invention also relates to a triparatopic amino acid sequence, Nanobody or polypeptide of the invention or a composition comprising the same that combines two or three different modes of action each mediated by one of the binding units of the triparatopic amino acid sequence, Nanobody or polypeptide of the invention, wherein each binding unit binds at a different binding site of HER2.

More generally, the present invention relates to a multiparatopic amino acid sequence, Nanobody or polypeptide of the invention or a composition comprising the same that combines two or more different modes of action each mediated by one of the binding units of the multiparatopic amino acid sequence, Nanobody or polypeptide of the invention, wherein each binding unit binds at a different binding site of HER2.

The invention further relates to methods for preparing or generating the amino acid sequences, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;     and -   b) screening said set, collection or library of amino acid sequences     for amino acid sequences that can bind to and/or have affinity for     HER2;     and -   c) isolating the amino acid sequence(s) that can bind to and/or have     affinity for HER2.

In such a method, the set, collection or library of amino acid sequences may be any suitable set, collection or library of amino acid sequences. For example, the set, collection or library of amino acid sequences may be a set, collection or library of immunoglobulin sequences (as described herein), such as a naïve set, collection or library of immunoglobulin sequences; a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of amino acid sequences may be a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of amino acid sequences may be a set, collection or library of domain antibodies or single domain antibodies, or may be a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of immunoglobulin sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In the above step b), the set, collection or library may for example be screened for (nucleic acid sequences that encode) amino acid sequences that can bind to the Herceptin® binding site on HER-2 (and may in particular to domain IV of HER2, more in particular to the C-terminus of domain IV of HER2) and/or that compete with Herceptin® for binding to HER-2.

Alternatively, in the above step b), the set, collection or library may for example be screened for (nucleic acid sequences that encode) amino acid sequences that can bind to the Omnitarg binding site on HER-2 (and may in particular to domain II of HER2, more in particular to the middle of domain II of HER2) and/or that compete with Omnitarg for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequences that encode) amino acid sequences that compete with Herceptin® or Omnitarg, respectively, may be performed using generally known methods for screening or selecting for competitors of known binding molecules, which may for example involve performing the screening or selection in the presence of the binding molecule and/or determining the binding affinity of the compound(s) to be screened in the presence of the binding molecule.

In another aspect, the method for generating amino acid sequences comprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid     sequences; -   b) screening said collection or sample of cells for cells that     express an amino acid sequence that can bind to and/or have affinity     for HER2;     and -   c) either (i) isolating said amino acid sequence; or (ii) isolating     from said cell a nucleic acid sequence that encodes said amino acid     sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulin sequence, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820 (2001).

Again, in the above step b), the set, collection or library may for example be screened for (nucleic acid sequences that encode) amino acid sequences that can bind to the Herceptin® binding site on HER-2 (and may in particular to domain IV of HER2, more in particular to the C-terminus of domain IV of HER2) and/or that compete with Herceptin® for binding to HER-2; or alternatively for (nucleic acid sequences that encode) amino acid sequences that can bind to the Omnitarg binding site on HER-2 (and may in particular to domain II of HER2, more in particular to the middle of domain II of HER2) and/or that compete with Omnitarg for binding to HER-2.

In another aspect, the method for generating an amino acid sequence directed against HER2 may comprise at least the steps of

-   a) providing a set, collection or library of nucleic acid sequences     encoding amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for HER2;     and -   c) isolating said nucleic acid sequence, followed by expressing said     amino acid sequence.

In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

Again, in the above step b), the set, collection or library may for example be screened for (nucleic acid sequences that encode) amino acid sequences that can bind to the Herceptin® binding site on HER-2 (and may in particular to domain IV of HER2, more in particular to the C-terminus of domain IV of HER2) and/or that compete with Herceptin® for binding to HER-2; or alternatively for (nucleic acid sequences that encode) amino acid sequences that can bind to the Omnitarg binding site on HER-2 (and may in particular to domain II of HER2, more in particular to the middle of domain II of HER2) and/or that compete with Omnitarg for binding to HER-2.

In another aspect, the method for generating an amino acid sequence directed against HER2 may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences     encoding amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for HER2 and that is     cross-blocked or is cross blocking a Nanobody of the invention, e.g.     SEQ ID NO: 2051-2325, or a polypeptide or construct of the     invention, e.g. SEQ ID NO: 2326-2390; and -   c) isolating said nucleic acid sequence, followed by expressing said     amino acid sequence.

In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 binding amino acid sequence fused to the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in. Nature Biotechnology, 23, 9, 1105-1116 (2005).

Also encompassed within the present invention are methods for preparing and generating multiparatopic (such as e.g. biparatopic, triparatopic, etc.) amino acids of the invention.

Without being limiting, a method for preparing and generating biparatopic amino acids of the invention may comprise at least the steps of:

-   a) providing a nucleic acid sequence encoding an HER2 binding amino     acid sequence fused to a set, collection or library of nucleic acid     sequences encoding amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for an antigenic     determinant on HER2 different from the antigenic determinant     recognized by the HER2 binding amino acid sequence;     and -   c) isolating the nucleic acid sequence encoding an HER2 binding     amino acid sequence fused to the nucleic acid sequence obtained in     b), followed by expressing the encoded amino acid sequence.

The biparatopic amino acid sequence obtained in the method above, can subsequently be fused to one or more further sets, collections or libraries of nucleic acid sequences encoding amino acid sequences and again screened for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for an antigenic determinant on HER2 different from the antigenic determinant of the HER2 binding amino acid sequence and the antigenic determinant of b) in order to obtain a triparatopic or multiparatopic amino acid sequence respectively.

in such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 binding amino acid sequence fused to the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

According to a particularly preferred aspect, a method for preparing and generating biparatopic amino acids of the invention may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences,     in which each nucleic acid sequence in said set, collection or     library encodes a fusion protein that comprises a first amino acid     sequence that can bind to and/or has affinity for a first antigenic     determinant, part, domain or epitope on HER2 that is fused     (optionally via a linker sequence) to a second amino acid sequence,     in which essentially each second amino acid sequence (or most of     these) is a different member of a set, collection or library of     different amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for a second antigenic     determinant, part, domain or epitope on HER2 different from the     first antigenic determinant, part, domain or epitope on HER-2;     and -   c) isolating the nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for a second antigenic     determinant, part, domain or epitope on HER2 different from the     first antigenic determinant, part, domain or epitope on HER-2,     obtained in b), optionally followed by expressing the encoded amino     acid sequence.

In this preferred method, the first amino acid sequence in the fusion protein encoded by said set collection or library of nucleic acid sequences may be the same amino acid sequence for all members of the set, collection or library of nucleic acid sequences encoding the fusion protein; or the first amino acid sequence in the fusion protein encoded by said set collection or library of nucleic acid sequences may also be a member of a set collection or library of different amino acid sequences.

Again, in such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences that form part of the fusion protein may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 binding amino acid sequence fused to the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 11.05-1116 (2005).

In step b), the set, collection or library of nucleic acid sequences may also be screened for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for both the first antigenic determinant, part, domain or epitope on HER2 and the second antigenic determinant, part, domain or epitope on HER2. This may for example be performed in a subsequent steps (i.e. by in a first step screening or selecting for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for the second antigenic determinant, part, domain or epitope on HER2, and subsequently in a second step selecting or screening for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for the first antigenic determinant, part, domain or epitope on HER2; or visa versa) or in a single step (i.e. by simultaneously screening or selecting for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for both the first antigenic determinant, part, domain or epitope on HER2 and the second antigenic determinant, part, domain or epitope on HER2).

In a preferred aspect of the above method, the first amino acid sequence used in step a) is preferably such that (i) it can bind to and/or has affinity for the Herceptin® binding site on HER2 (and may in particular be directed against domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or (ii) competes with Herceptin® for binding to HER-2; and in step b), the set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode (i) an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or (ii) an amino acid sequence that can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2.

Alternatively, the first amino acid sequence used in step a) is preferably such that (i) it can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain IT of HER2, more in particular the middle of domain II of HER2) and/or (ii) competes with Omnitarg for binding to HER-2; and in step b), the set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode (i) an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or (ii) an amino acid sequence that can compete with Herceptin® for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequences that encode) amino acid sequences that compete with Herceptin® or Omnitarg, respectively, may be performed using generally known methods for screening or selecting for competitors of known binding molecules, which may for example involve performing the screening or selection in the presence of the binding molecule and/or determining the binding affinity of the compound(s) to be screened in the presence of the binding molecule.

It is also possible, in step b), to screen for nucleic acid sequences that both (i) encode an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or that can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2; and that also (ii) encode an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or that can compete with Herceptin® for binding to HER-2. Again, this may be performed in separate steps or a single step, and by selecting or screening in the presence of Herceptin® and/or Omnitarg, as applicable.

It will also be clear to the skilled person that the above methods may be performed by screening a set, collection or library of amino acid sequences that correspond to (e.g. are encoded by) the nucleic acid sequences used in the above method; and such methods form further aspects of the invention.

The invention in a further aspect provides a method for preparing and generating biparatopic amino acids of the invention which comprises at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences,     in which each nucleic acid sequence in said set, collection or     library encodes a fusion protein that comprises a first amino acid     sequence that can bind to and/or has affinity for a first antigenic     determinant, part, domain or epitope on HER2 that is fused via a     linker sequence to a second amino acid sequence that can bind to     and/or has affinity for a second antigenic determinant, part, domain     or epitope on HER2 (which may be the same or different as the first     antigenic determinant, part, domain or epitope on HER2), in which     essentially each nucleic acid sequence (or most of these) encodes a     fusion protein with a different linker sequence so as to provide a     set, collection or library of nucleic acid sequences encoding     different fusion proteins; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for the first and     second antigenic determinant, part, domain or epitope on HER2;     and -   c) isolating the nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for the first and     second antigenic determinant, part, domain or epitope on HER2,     optionally followed by expressing the encoded amino acid sequence.

As will be clear to the skilled person, this method can be used to screen for suitable or even optimal linker lengths for linking the first and second amino acid sequence. For example, in this aspect, the first amino acid sequence may be an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or that can compete with Omnitarg (or the Omnitarg Fab used in Example 9); and the second amino acid sequence may be an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or that can compete with Herceptin® for binding to HER-2 (or visa versa). The screening and selection step b) may be performed as further described above.

Another method for preparing and generating biparatopic amino acids of the invention may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences     encoding amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for a set, collection or library of nucleic acid sequences     that encode an amino acid sequence that can bind to and/or has     affinity for HER2; -   c) ligating said set, collection or library of nucleic acid     sequences that encode an amino acid sequence that can bind to and/or     has affinity for HER2 to another nucleic acid sequence that encodes     an amino acid sequence that can bind to and/or has affinity for HER2     (e.g. a nucleic acid sequence that encodes an amino acid sequence     that competes with Herceptin® for binding HER2);     and -   d) from the set, collection or library of nucleic acid sequences     obtained in c), isolating the nucleic acid sequences encoding a     biparatopic amino acid sequence that can bind to and/or has affinity     for HER2 (and e.g. further selecting for nucleic acid sequences that     encode a biparatopic amino acid sequence that antagonizes with     higher potency compared to the monovalent amino acid sequences),     followed by expressing the encoded amino acid sequence.

The biparatopic amino acid sequence obtained in the method above, can subsequently be fused to one or more further sets, collections or libraries of nucleic acid sequences encoding amino acid sequences that can bind to and/or have affinity for HER2 in order to obtain a triparatopic or multiparatopic amino acid sequence respectively.

In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

The set, collection or library of nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for HER2 can be obtained by any selection or screening method known in the art for the selection and/or screening of nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for HER2 and as, for example, described in the Examples section.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

Another method for preparing and generating biparatopic amino acids of the invention may comprise at least the steps of:

-   a) providing a first set, collection or library of nucleic acid     sequences encoding amino acid sequences; -   b) screening said first set, collection or library of nucleic acid     sequences for a nucleic acid sequence that encodes an amino acid     sequence that can bind to and/or has affinity for a first antigenic     determinant, part, domain or epitope on HER2; -   c) ligating the nucleic acid sequence encoding said amino acid     sequence that can bind to and/or has affinity for a first antigenic     determinant, part, domain or epitope on HER2 obtained in b) to     another set, collection or library of nucleic acid sequences     encoding amino acid sequences to obtain a set, collection or library     of nucleic acid sequences that encode fusion proteins; -   d) screening said set, collection or library of nucleic acid     sequences obtained in step c) for a nucleic acid sequence that     encodes an amino acid sequence that can bind a second antigenic     determinant, part, domain or epitope on HER2 different from the     first antigenic determinant, part, domain or epitope on HER-2;     and -   e) isolating the nucleic acid sequence that encodes an amino acid     sequence that can bind to and/or has affinity for the first and     second antigenic determinant, part, domain or epitope on HER2,     optionally followed by expressing the encoded amino acid sequence.

In a preferred aspect of the above method, the first amino acid sequence obtained in step b) is preferably such that (i) it can bind to and/or has affinity for the Herceptin® binding site on HER2 (and may in particular be directed against domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or (ii) competes with Herceptin® for binding to HER-2; and in step d), the set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode (i) an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or (ii) an amino acid sequence that can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2.

Alternatively, the first amino acid sequence obtained in step b) is preferably such that (i) it can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or (ii) competes with Omnitarg for binding to HER-2; and in step d), the set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode (i) an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or (ii) an amino acid sequence that can compete with Herceptin® for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequences that encode) amino acid sequences that compete with Herceptin® or Omnitarg, respectively, may be performed using generally known methods for screening or selecting for competitors of known binding molecules, which may for example involve performing the screening or selection in the presence of the binding molecule and/or determining the binding affinity of the compound(s) to be screened in the presence of the binding molecule.

It is also possible, in step d), to screen for nucleic acid sequences that both (i) encode an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or that can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2; and that also (ii) encode an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or that can compete with Herceptin® for binding to HER-2. Again, this may be performed in separate steps or a single step, and by selecting or screening in the presence of Herceptin® and/or Omnitarg, as applicable.

The biparatopic amino acid sequence obtained in the method above, can subsequently be fused to one or more further sets, collections or libraries of nucleic acid sequences encoding amino acid sequences that can bind to and/or have affinity for HER2 in order to obtain a triparatopic or multiparatopic amino acid sequence respectively. In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

The set, collection or library of nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for HER2 can be obtained by any selection or screening method known in the art for the selection and/or screening of nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for HER2 and as, for example, described in the Examples section.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

The invention also relates to amino acid sequences that are obtained by the above methods, or alternatively by a method that comprises one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said immunoglobulin sequence; and of expressing or synthesizing said amino acid sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.

Another method for preparing multivalent and/or multiparatopic/biparatopic amino acids or constructs of the invention may comprise at least the steps of linking two or more monovalent amino acid sequences or monovalent construct of the invention and for example one or more linkers together in a suitable manner. The monovalent constructs (and linkers) can be coupled by any method known in the art and as further described herein. Preferred techniques include the linking of the nucleic acid sequences that encode the monovalent constructs (and linkers) to prepare a genetic construct that expresses the multivalent and/or multiparatopic/biparatopic amino acid or construct. Techniques for linking amino acid sequences or nucleic acid sequences will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as the Examples below.

Accordingly, the present invention also relates to the use of a monovalent construct (which may comprise or essentially consists of an amino acid sequence of the invention such as a domain antibody, an amino acid sequence that is suitable for use as a domain antibody, a single domain antibody, an amino acid sequence that is suitable for use as a single domain antibody, a “dAb”, an amino acid sequences that is suitable for use as a dAb, or a Nanobody) in providing and/or preparing a multivalent (such as multiparatopic, and preferably biparatopic) compound or construct. The monovalent construct is then used as a binding domain or binding unit in providing and/or preparing the multivalent (such as multiparatopic, and preferably biparatopic) construct comprising two (e.g. in a biparatopic construct) or more (e.g. in a multiparatopic construct) binding units. In this respect, the monovalent construct may be used as a binding domain or binding unit in providing and/or preparing a multivalent (such as multiparatopic, and preferably biparatopic) construct of the invention comprising two or more binding units.

In a preferred aspect, the monovalent construct (which may comprise or essentially consists of an amino acid sequence of the invention such as a domain antibody, an amino acid sequence that is suitable for use as a domain antibody, a single domain antibody, an amino acid sequence that is suitable for use as a single domain antibody, a “dAb”, an amino acid sequences that is suitable for use as a dAb, or a Nanobody) is used in providing and/or preparing a multivalent (such as multiparatopic, and preferably biparatopic) construct that exhibits intramolecular binding compared to intermolecular binding. In such multivalent constructs of the invention that comprises amino acid sequences directed against two or more (different) antigenic determinants on the same antigen (for example against different epitopes of an antigen and/or against different subunits of a multimeric receptor or protein), the length and flexibility of the linker are preferably such that, when the multivalent construct binds to HER-2, at least two and preferably all of the amino acid sequences that are present in the multivalent construct can (simultaneously) bind to each of their intended antigenic determinants, epitopes, parts or domains, most preferably so as to allow binding with increased avidity and also intramolecular binding and/or recognition. Accordingly, the present invention also relates to the use of a monovalent construct (which may comprise or essentially consists of an amino acid sequence of the invention such as a domain antibody, an amino acid sequence that is suitable for use as a domain antibody, a single domain antibody, an amino acid sequence that is suitable for use as a single domain antibody, a “dAb”, an amino acid sequences that is suitable for use as a dAb, or a Nanobody) as a binding domain or binding unit in providing and/or preparing a multivalent (such as multiparatopic, and preferably biparatopic) construct, wherein the binding domains or binding units are linked via a linker such that the multivalent (such as multiparatopic, and preferably biparatopic) construct preferably exhibits intramolecular binding compared to intermolecular binding.

In some of the most preferred multiparatopic polypeptides of the invention, (i) at least one monovalent construct of the invention (and in particular at least one Nanobody) is used that is directed against the Omnitarg binding site on HER2 (and in particular against domain II of HER2, and more in particular against the middle of domain II of HER2) and/or that is capable of competing with Omnitarg for binding to HER-2; and at least one amino acid sequence of the invention (and in particular at least one Nanobody) is used that is directed against another antigenic determinant, epitope, part or domain of HER2. In such a preferred multiparatopic construct of the invention, the linker is most preferably such that the multiparatopic construct of the invention is capable of (simultaneously) binding to both the Omnitarg binding site on HER2 (and in particular against domain II of HER2, and more in particular against the middle of domain II of HER2) as well as the other antigenic determinant, epitope, part or domain of HER2, again most preferably so as to allow binding with increased avidity and also intramolecular binding and/or recognition. Accordingly, also encompassed in the present invention is the use of a monovalent construct comprising an amino acid of the invention (and in particular a Nanobody) that is directed against the Omnitarg binding site on HER2 (and in particular against domain II of HER2, and more in particular against the middle of domain II of HER2) and/or that is capable of competing with Omnitarg for binding to HER-2, as a binding domain or binding unit in providing and/or preparing a multiparatopic (such as biparatopic) construct, wherein the binding domains or binding units are linked via a linker such that the multiparatopic (such as biparatopic) construct preferably exhibits intramolecular binding compared to intermolecular binding.

In some of the most preferred multiparatopic polypeptides of the invention, (i) at least one monovalent construct of the invention (and in particular at least one Nanobody) is used that is directed against the Herceptin® binding site on HER2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2) and/or that is capable of competing with Herceptin® for binding to HER-2; and at least one amino acid of the invention (and in particular at least one Nanobody) is used that is directed against another antigenic determinant, epitope, part or domain of HER2. In such a preferred multiparatopic construct of the invention, the linker is most preferably such that the multiparatopic construct of the invention is capable of (simultaneously) binding to both the Herceptin® binding site on HER2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2), as well as the other antigenic determinant, epitope, part or domain of HER2, again most preferably so as to allow binding with increased avidity and also intramolecular binding and/or recognition. Accordingly, also encompassed in the present invention is the use of a monovalent construct comprising an amino acid sequence of the invention (and in particular at least one Nanobody) that is directed against the Herceptin® binding site on HER2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2) and/or that is capable of competing with Herceptin® for binding to HER-2, as a binding domain or binding unit in providing and/or preparing a multiparatopic (such as a biparatopic) construct, wherein the binding domains or binding units are linked via a linker such that the multiparatopic (such as biparatopic) construct preferably exhibits intramolecular binding compared to intermolecular binding.

In some of the most preferred multiparatopic polypeptides of the invention, (i) at least one monovalent construct of the invention (and in particular at least one Nanobody) is used that is directed against the Omnitarg binding site on HER2 (and in particular against domain II of HER2, and more in particular against the middle of domain II of HER2) and/or that is capable of competing with Omnitarg for binding to HER-2; and at least one monovalent construct of the invention (and in particular at least one Nanobody) is used that is directed against the Herceptin® binding site on HER2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2) and/or that is capable of competing with Herceptin® for binding to HER-2. In such a preferred multiparatopic construct of the invention, the linker is most preferably such that the multiparatopic construct of the invention is capable of (simultaneously) binding to both the Omnitarg binding site on HER2 (and in particular against domain II of HER2, and more in particular against the middle of domain II of HER2) as well as the Herceptin® binding site on HER2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2), again most preferably so as to allow binding with increased avidity and also intramolecular binding and/or recognition. Accordingly, also encompassed in the present invention is the use of a monovalent construct comprising an amino acid sequence of the invention (and in particular at least one Nanobody) that is directed against the Herceptin® binding site on HER2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2) and/or that is capable of competing with Herceptin® for binding to HER-2, and a monovalent construct comprising an amino acid of the invention (and in particular a Nanobody) that is directed against the Omnitarg binding site on HER2 (and in particular against domain II of HER2, and more in particular against the middle of domain II of HER2) and/or that is capable of competing with Omnitarg for binding to HER-2, as binding domains or binding units in providing and/or preparing a multiparatopic (such as a biparatopic) construct, wherein the binding domains or binding units are linked via a linker such that the multiparatopic (such as biparatopic) construct preferably exhibits intramolecular binding compared to intermolecular binding.

The invention also relates to amino acid sequences that are obtained by the above methods, or alternatively by a method that comprises one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said immunoglobulin sequence; and of expressing or synthesizing said amino acid sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.

In this respect, the present invention also relates to the use of a nucleic acid or nucleotide sequence that encodes a monovalent construct of the invention for the preparation of a genetic construct (as further defined herein) that encodes a multivalent (such as multiparatopic, and preferably biparatopic) construct. Also, as will be clear to the skilled person, to prepare such a genetic construct, encoding a multivalent (such as multiparatopic, and preferably biparatopic) construct of the invention, several nucleotide sequences, such as at least two nucleotide sequences encoding a monovalent construct of the invention and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner. Such genetic constructs generally also comprises one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein.

Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more “mismatched” primers. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as the Examples below.

Also, following the steps above, one or more amino acid sequences of the invention may be suitably humanized (or alternatively camelized); and/or the amino acid sequence(s) thus obtained may be linked to each other or to one or more other suitable amino acid sequences (optionally via one or more suitable linkers) so as to provide a polypeptide of the invention. Also, a nucleic acid sequence encoding an amino acid sequence of the invention may be suitably humanized (or alternatively camelized) and suitably expressed; and/or one or more nucleic acid sequences encoding an amino acid sequence of the invention may be linked to each other or to one or more nucleic acid sequences that encode other suitable amino acid sequences (optionally via nucleotide sequences that encode one or more suitable linkers), after which the nucleotide sequence thus obtained may be suitably expressed so as to provide a polypeptide of the invention.

The invention further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with HER2. Some preferred but non-limiting applications and uses will become clear from the further description herein.

The invention also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy.

In particular, the invention also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of a disease or disorder that can be prevented or treated by administering, to a subject in need thereof, of (a pharmaceutically effective amount of) an amino acid sequence, compound, construct or polypeptide as described herein.

More in particular, the invention relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of cancers and/or tumors.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description herein, in which the invention will be described and discussed in more detail with reference to the Nanobodies of the invention and polypeptides of the invention comprising the same, which form some of the preferred aspects of the invention.

As will become clear from the further description herein, Nanobodies generally offer certain advantages (outlined herein) compared to “dAb's” or similar (single) domain antibodies or immunoglobulin sequences, which advantages are also provided by the Nanobodies of the invention. However, it will be clear to the skilled person that the more general aspects of the teaching below can also be applied (either directly or analogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their     usual meaning in the art, which will be clear to the skilled person.     Reference is for example made to the standard handbooks, such as     Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd. Ed.),     Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et     al, eds., “Current protocols in molecular biology”, Green Publishing     and Wiley Interscience, New York (1987); Lewin, “Genes II”, John     Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of     Gene Manipulation: An Introduction to Genetic Engineering”, 2nd     edition, University of California Press, Berkeley, Calif. (1981);     Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh     (2001); Roitt et al., Roitt's Essential Immunology, 10^(th) Ed.     Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology”     (6th Ed.), Garland Science Publishing/Churchill Livingstone, N.Y.     (2005), as well as to the general background art cited herein; -   b) Unless indicated otherwise, the term “immunoglobulin     sequence”—whether used herein to refer to a heavy chain antibody or     to a conventional 4-chain antibody—is used as a general term to     include both the full-size antibody, the individual chains thereof,     as well as all parts, domains or fragments thereof (including but     not limited to antigen-binding domains or fragments such as V_(HH)     domains or V_(H)/V_(L) domains, respectively). In addition, the term     “sequence” as used herein (for example in terms like “immunoglobulin     sequence”, “antibody sequence”, “variable domain sequence”, “V_(HH)     sequence” or “protein sequence”), should generally be understood to     include both the relevant amino acid sequence as well as nucleic     acids or nucleotide sequences encoding the same, unless the context     requires a more limited interpretation. Also, the term “nucleotide     sequence” as used herein also encompasses a nucleic acid molecule     with said nucleotide sequence, so that the terms “nucleotide     sequence” and “nucleic acid” should be considered equivalent and are     used interchangeably herein; -   c) Unless indicated otherwise, all methods, steps, techniques and     manipulations that are not specifically described in detail can be     performed and have been performed in a manner known per se, as will     be clear to the skilled person. Reference is for example again made     to the standard handbooks and the general background art mentioned     herein and to the further references cited therein; as well as to     for example the following reviews Presta, Adv. Drug Deliv. Rev.     2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):     49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;     Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et     al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for     protein engineering, such as affinity maturation and other     techniques for improving the specificity and other desired     properties of proteins such as immunoglobulins. -   d) Amino acid residues will be indicated according to the standard     three-letter or one-letter amino acid code, as mentioned in Table     A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, Alanine Ala A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L Isoleucine Ile I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W Proline Pro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0) Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q Tyrosine Tyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0) Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimes also considered to be a polar uncharged amino acid. ⁽²⁾Sometimes also considered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear to the skilled person, the fact that an amino acid residue is referred to in this Table as being either charged or uncharged at pH 6.0 to 7.0 does not reflect in any way on the charge said amino acid residue may have at a pH lower than 6.0 and/or at a pH higher than 7.0; the amino acid residues mentioned in the Table can be either charged and/or uncharged at such a higher or lower pH, as will be clear to the skilled person. ⁽⁴⁾As is known in the art, the charge of a His residue is greatly dependant upon even small shifts in pH, but a His residu can generally be considered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,     the percentage of “sequence identity” between a first nucleotide     sequence and a second nucleotide sequence may be calculated by     dividing [the number of nucleotides in the first nucleotide sequence     that are identical to the nucleotides at the corresponding positions     in the second nucleotide sequence] by [the total number of     nucleotides in the first nucleotide sequence] and multiplying by     [100%], in which each deletion, insertion, substitution or addition     of a nucleotide in the second nucleotide sequence—compared to the     first nucleotide sequence—is considered as a difference at a single     nucleotide (position). -    Alternatively, the degree of sequence identity between two or more     nucleotide sequences may be calculated using a known computer     algorithm for sequence alignment such as NCBI Blast v2.0, using     standard settings. -    Some other techniques, computer algorithms and settings for     determining the degree of sequence identity are for example     described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318,     WO 00/78972, WO 98/49185 and GB 2 357 768-A. -    Usually, for the purpose of determining the percentage of “sequence     identity” between two nucleotide sequences in accordance with the     calculation method outlined hereinabove, the nucleotide sequence     with the greatest number of nucleotides will be taken as the “first”     nucleotide sequence, and the other nucleotide sequence will be taken     as the “second” nucleotide sequence; -   f) For the purposes of comparing two or more amino acid sequences,     the percentage of “sequence identity” between a first amino acid     sequence and a second amino acid sequence (also referred to herein     as “amino acid identity”) may be calculated by dividing [the number     of amino acid residues in the first amino acid sequence that are     identical to the amino acid residues at the corresponding positions     in the second amino acid sequence] by [the total number of amino     acid residues in the first amino acid sequence] and multiplying by     [100%], in which each deletion, insertion, substitution or addition     of an amino acid residue in the second amino acid sequence—compared     to the first amino acid sequence—is considered as a difference at a     single amino acid residue (position), i.e. as an “amino acid     difference” as defined herein. -    Alternatively, the degree of sequence identity between two amino     acid sequences may be calculated using a known computer algorithm,     such as those mentioned above for determining the degree of sequence     identity for nucleotide sequences, again using standard settings. -    Usually, for the purpose of determining the percentage of “sequence     identity” between two amino acid sequences in accordance with the     calculation method outlined hereinabove, the amino acid sequence     with the greatest number of amino acid residues will be taken as the     “first” amino acid sequence, and the other amino acid sequence will     be taken as the “second” amino acid sequence. -    Also, in determining the degree of sequence identity between two     amino acid sequences, the skilled person may take into account     so-called “conservative” amino acid substitutions, which can     generally be described as amino acid substitutions in which an amino     acid residue is replaced with another amino acid residue of similar     chemical structure and which has little or essentially no influence     on the function, activity or other biological properties of the     polypeptide. Such conservative amino acid substitutions are well     known in the art, for example from WO 04/037999, GB-A-3 357 768, WO     98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or     combinations of such substitutions may be selected on the basis of     the pertinent teachings from WO 04/037999 as well as WO 98/49185 and     from the further references cited therein. -    Such conservative substitutions preferably are substitutions in     which one amino acid within the following groups (a)-(e) is     substituted by another amino acid residue within the same group: (a)     small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,     Pro and Gly; (b) polar, negatively charged residues and their     (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively     charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar     residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues:     Phe, Tyr and Trp. -    Particularly preferred conservative substitutions are as follows:     Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His;     Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala     or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu     into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into     Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser     into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into     Val, into Ile or into Leu. -    Any amino acid substitutions applied to the polypeptides described     herein may also be based on the analysis of the frequencies of amino     acid variations between homologous proteins of different species     developed by Schulz et al., Principles of Protein Structure,     Springer-Verlag, 1978, on the analyses of structure forming     potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974     and Adv. Enzymol., 47: 45-149, 1978, and on the analysis of     hydrophobicity patterns in proteins developed by Eisenberg et al.,     Proc. Nad. Acad. Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J     Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev.     Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their     entirety by reference. Information on the primary, secondary and     tertiary structure of Nanobodies is given in the description herein     and in the general background art cited above. Also, for this     purpose, the crystal structure of a V_(HH) domain from a llama is     for example given by Desmyter et al., Nature Structural. Biology,     Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology     (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361     (1999). Further information about some of the amino acid residues     that in conventional V_(H) domains form the V_(H)/V_(L) interface     and potential camelizing substitutions on these positions can be     found in the prior art cited above. -   g) Amino acid sequences and nucleic acid sequences are said to be     “exactly the same” if they have 100% sequence identity (as defined     herein) over their entire length; -   h) When comparing two amino acid sequences, the term “amino acid     difference” refers to an insertion, deletion or substitution of a     single amino acid residue on a position of the first sequence,     compared to the second sequence; it being understood that two amino     acid sequences can contain one, two or more such amino acid     differences; -   i) When a nucleotide sequence or amino acid sequence is said to     “comprise” another nucleotide sequence or amino acid sequence,     respectively, or to “essentially consist of” another nucleotide     sequence or amino acid sequence, this may mean that the latter     nucleotide sequence or amino acid sequence has been incorporated     into the firstmentioned nucleotide sequence or amino acid sequence,     respectively, but more usually this generally means that the     firstmentioned nucleotide sequence or amino acid sequence comprises     within its sequence a stretch of nucleotides or amino acid residues,     respectively, that has the same nucleotide sequence or amino acid     sequence, respectively, as the latter sequence, irrespective of how     the firstmentioned sequence has actually been generated or obtained     (which may for example be by any suitable method described herein).     By means of a non-limiting example, when a Nanobody of the invention     is said to comprise a CDR sequence, this may mean that said CDR     sequence has been incorporated into the Nanobody of the invention,     but more usually this generally means that the Nanobody of the     invention contains within its sequence a stretch of amino acid     residues with the same amino acid sequence as said CDR sequence,     irrespective of how said Nanobody of the invention has been     generated or obtained. It should also be noted that when the latter     amino acid sequence has a specific biological or structural     function, it preferably has essentially the same, a similar or an     equivalent biological or structural function in the firstmentioned     amino acid sequence (in other words, the firstmentioned amino acid     sequence is preferably such that the latter sequence is capable of     performing essentially the same, a similar or an equivalent     biological or structural function). For example, when a Nanobody of     the invention is said to comprise a CDR sequence or framework     sequence, respectively, the CDR sequence and framework are     preferably capable, in said Nanobody, of functioning as a CDR     sequence or framework sequence, respectively. Also, when a     nucleotide sequence is said to comprise another nucleotide sequence,     the firstmentioned nucleotide sequence is preferably such that, when     it is expressed into an expression product (e.g. a polypeptide), the     amino acid sequence encoded by the latter nucleotide sequence forms     part of said expression product (in other words, that the latter     nucleotide sequence is in the same reading frame as the     firstmentioned, larger nucleotide sequence). -   j) A nucleic acid sequence or amino acid sequence is considered to     be “(in) essentially isolated (form)”—for example, compared to its     native biological source and/or the reaction medium or cultivation     medium from which it has been obtained—when it has been separated     from at least one other component with which it is usually     associated in said source or medium, such as another nucleic acid,     another protein/polypeptide, another biological component or     macromolecule or at least one contaminant, impurity or minor     component. In particular, a nucleic acid sequence or amino acid     sequence is considered “essentially isolated” when it has been     purified at least 2-fold, in particular at least 10-fold, more in     particular at least 100-fold, and up to 1000-fold or more. A nucleic     acid sequence or amino acid sequence that is “in essentially     isolated form” is preferably essentially homogeneous, as determined     using a suitable technique, such as a suitable chromatographical     technique, such as polyacrylamide-gel electrophoresis; -   k) The term “domain” as used herein generally refers to a globular     region of an amino acid sequence (such as an antibody chain, and in     particular to a globular region of a heavy chain antibody), or to a     polypeptide that essentially consists of such a globular region.     Usually, such a domain will comprise peptide loops (for example 3 or     4 peptide loops) stabilized, for example, as a sheet or by disulfide     bonds. The term “binding domain” refers to such a domain that is     directed against an antigenic determinant (as defined herein); -   l) The term “antigenic determinant” refers to the epitope on the     antigen recognized by the antigen-binding molecule (such as a     Nanobody or a polypeptide of the invention) and more in particular     by the antigen-binding site of said molecule. The terms “antigenic     determinant” and “epitope” may also be used interchangeably herein. -   m) An amino acid sequence (such as a Nanobody, an antibody, a     polypeptide of the invention, or generally an antigen binding     protein or polypeptide or a fragment thereof) that can     (specifically) bind to, that has affinity for and/or that has     specificity for a specific antigenic determinant, epitope, antigen     or protein (or for at least one part, fragment or epitope thereof)     is said to be “against” or “directed against” said antigenic     determinant, epitope, antigen or protein. -   n) The term “specificity” refers to the number of different types of     antigens or antigenic determinants to which a particular     antigen-binding molecule or antigen-binding protein (such as a     Nanobody or a polypeptide of the invention) molecule can bind. The     specificity of an antigen-binding protein can be determined based on     affinity and/or avidity. The affinity, represented by the     equilibrium constant for the dissociation of an antigen with an     antigen-binding protein (K_(D)), is a measure for the binding     strength between an antigenic determinant and an antigen-binding     site on the antigen-binding protein: the lesser the value of the     K_(D), the stronger the binding strength between an antigenic     determinant and the antigen-binding molecule (alternatively, the     affinity can also be expressed as the affinity constant (K_(A)),     which is 1/K_(D)). As will be clear to the skilled person (for     example on the basis of the further disclosure herein), affinity can     be determined in a manner known per se, depending on the specific     antigen of interest. Avidity is the measure of the strength of     binding between an antigen-binding molecule (such as a Nanobody or     polypeptide of the invention) and the pertinent antigen. Avidity is     related to both the affinity between an antigenic determinant and     its antigen binding site on the antigen-binding molecule and the     number of pertinent binding sites present on the antigen-binding     molecule. Typically, antigen-binding proteins (such as the amino     acid sequences, Nanobodies and/or polypeptides of the invention)     will bind to their antigen with a dissociation constant (K_(D)) of     10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²     moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter     (i.e. with an association constant (K_(A)) of 10⁵ to 10¹²     liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more     and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value     greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)     liters/mol is generally considered to indicate non-specific binding.     Preferably, a monovalent immunoglobulin sequence of the invention     will bind to the desired antigen with an affinity less than 500 nM,     preferably less than 200 nM, more preferably less than 10 nM, such     as less than 500 pM. Specific binding of an antigen-binding protein     to an antigen or antigenic determinant can be determined in any     suitable manner known per se, including, for example, Scatchard     analysis and/or competitive binding assays, such as     radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich     competition assays, and the different variants thereof known per se     in the art; as well as the other techniques mentioned herein. -    The dissociation constant may be the actual or apparent     dissociation constant, as will be clear to the skilled person.     Methods for determining the dissociation constant will be clear to     the skilled person, and for example include the techniques mentioned     herein. In this respect, it will also be clear that it may not be     possible to measure dissociation constants of more then 10⁻⁴     moles/liter or 10⁻³ moles/liter (e.g. of 10⁻² moles/liter).     Optionally, as will also be clear to the skilled person, the (actual     or apparent) dissociation constant may be calculated on the basis of     the (actual or apparent) association constant (K_(A)), by means of     the relationship [K_(D)=1/K_(A)]. -    The affinity denotes the strength or stability of a molecular     interaction. The affinity is commonly given as by the K_(D), or     dissociation constant, which has units of mol/liter (or M). The     affinity can also be expressed as an association constant, K_(A),     which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or M⁻¹). In the     present specification, the stability of the interaction between two     molecules (such as an amino acid sequence, Nanobody or polypeptide     of the invention and its intended target) will mainly be expressed     in terms of the K_(D) value of their interaction; it being clear to     the skilled person that in view of the relation K_(A)=1/K_(D),     specifying the strength of molecular interaction by its K_(D) value     can also be used to calculate the corresponding K_(A) value. The     K_(D)-value characterizes the strength of a molecular interaction     also in a thermodynamic sense as it is related to the free energy     (DG) of binding by the well known relation DG=RT·ln(K_(D))     (equivalently DG=−RT·ln(K_(A))), where R equals the gas constant, T     equals the absolute temperature and ln denotes the natural     logarithm. -    The K_(D) for biological interactions which are considered     meaningful (e.g. specific) are typically in the range of 10⁻¹⁰M (0.1     nM) to 10⁻⁵M (10000 nM). The stronger an interaction is, the lower     is its K_(D). -    The K_(D) can also be expressed as the ratio of the dissociation     rate constant of a complex, denoted as k_(off), to the rate of its     association, denoted k_(on) (so that K_(D)=k_(off)/k_(on) and     K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s     is the SI unit notation of second). The on-rate k_(on) has units M⁻¹     s⁻¹. The on-rate may vary between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,     approaching the diffusion-limited association rate constant for     bimolecular interactions. The off-rate is related to the half-life     of a given molecular interaction by the relation     t_(1/2)=ln(2)/k_(off). The off-rate may vary between 10⁻⁶ s⁻¹ (near     irreversible complex with a t_(1/2) of multiple days) to 1 s⁻¹     (t_(1/2)=0.69 s). -    The affinity of a molecular interaction between two molecules can     be measured via different techniques known per se, such as the well     known surface plasmon resonance (SPR) biosensor technique (see for     example Ober et al., Intern. Immunology, 13, 1551-1559, 2001) where     one molecule is immobilized on the biosensor chip and the other     molecule is passed over the immobilized molecule under flow     conditions yielding k_(on), k_(off) measurements and hence K_(D) (or     K_(A)) values. This can for example be performed using the     well-known BIACORE instruments. -    It will also be clear to the skilled person that the measured K_(D)     may correspond to the apparent K_(D) if the measuring process     somehow influences the intrinsic binding affinity of the implied     molecules for example by artefacts related to the coating on the     biosensor of one molecule. Also, an apparent K_(D) may be measured     if one molecule contains more than one recognition sites for the     other molecule. In such situation the measured affinity may be     affected by the avidity of the interaction by the two molecules. -    Another approach that may be used to assess affinity is the 2-step     ELISA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et     al. (J. Immunol. Methods, 77, 305-19, 1985). This method establishes     a solution phase binding equilibrium measurement and avoids possible     artefacts relating to adsorption of one of the molecules on a     support such as plastic. -    However, the accurate measurement of K_(D) may be quite     labor-intensive and as consequence, often apparent K_(D) values are     determined to assess the binding strength of two molecules. It     should be noted that as long all measurements are made in a     consistent way (e.g. keeping the assay conditions unchanged)     apparent K_(D) measurements can be used as an approximation of the     true K_(D) and hence in the present document K_(D) and apparent     K_(D) should be treated with equal importance or relevance. Finally,     it should be noted that in many situations the experienced scientist     may judge it to be convenient to determine the binding affinity     relative to some reference molecule. For example, to assess the     binding strength between molecules A and B, one may e.g. use a     reference molecule C that is known to bind to B and that is suitably     labelled with a fluorophore or chromophore group or other chemical     moiety, such as biotin for easy detection in an ELISA or FACS     (Fluorescent activated cell sorting) or other format (the     fluorophore for fluorescence detection, the chromophore for light     absorption detection, the biotin for streptavidin-mediated ELISA     detection). Typically, the reference molecule C is kept at a fixed     concentration and the concentration of A is varied for a given     concentration or amount of B. As a result an IC₅₀ value is obtained     corresponding to the concentration of A at which the signal measured     for C in absence of A is halved. Provided K_(D ref), the K_(D) of     the reference molecule, is known, as well as the total concentration     c_(ref) of the reference molecule, the apparent K_(D) for the     interaction A-B can be obtained from following formula:     K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if c_(ref)<<K_(D ref),     K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in a     consistent way (e.g. keeping c_(ref) fixed) for the binders that are     compared, the strength or stability of a molecular interaction can     be assessed by the IC₅₀ and this measurement is judged as equivalent     to K_(D) or to apparent K_(D) throughout this text. -   o) The half-life of an amino acid sequence, compound or polypeptide     of the invention can generally be defined as the time taken for the     serum concentration of the amino acid sequence, compound or     polypeptide to be reduced by 50%, in vivo, for example due to     degradation of the sequence or compound and/or clearance or     sequestration of the sequence or compound by natural mechanisms. The     in vivo half-life of an amino acid sequence, compound or polypeptide     of the invention can be determined in any manner known per se, such     as by pharmacokinetic analysis. Suitable techniques will be clear to     the person skilled in the art, and may for example generally involve     the steps of suitably administering to a warm-blooded animal (i.e.     to a human or to another suitable mammal, such as a mouse, rabbit,     rat, pig, dog or a primate, for example monkeys from the genus     Macaca (such as, and in particular, cynomologus monkeys (Macaca     fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon     (Papio ursinus)) a suitable dose of the amino acid sequence,     compound or polypeptide of the invention; collecting blood samples     or other samples from said animal; determining the level or     concentration of the amino acid sequence, compound or polypeptide of     the invention in said blood sample; and calculating, from (a plot     of) the data thus obtained, the time until the level or     concentration of the amino acid sequence, compound or polypeptide of     the invention has been reduced by 50% compared to the initial level     upon dosing. Reference is for example made to the Experimental Part     below, as well as to the standard handbooks, such as Kenneth, A et     al: Chemical Stability of Pharmaceuticals: A Handbook for     Pharmacists and Peters et al, Pharmacokinete analysis: A Practical     Approach (1996). Reference is also made to “Pharmacokinetics”, M     Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition     (1982). -    As will also be clear to the skilled person (see for example pages     6 and 7 of WO 04/003019 and in the further references cited     therein), the half-life can be expressed using parameters such as     the t1/2-alpha, t1/2-beta and the area under the curve (AUC). In the     present specification, an “increase in half-life” refers to an     increase in any one of these parameters, such as any two of these     parameters, or essentially all three these parameters. As used     herein “increase in half-life” or “increased half-life” in     particular refers to an increase in the t1/2-beta, either with or     without an increase in the t1/2-alpha and/or the AUC or both. -   p) In the context of the present invention, “modulating” or “to     modulate” generally means either reducing or inhibiting the activity     of, or alternatively increasing the activity of, a target or     antigen, as measured using a suitable in vitro, cellular or in vivo     assay. In particular, “modulating” or “to modulate” may mean either     reducing or inhibiting the activity of, or alternatively increasing     a (relevant or intended) biological activity of, a target or     antigen, as measured using a suitable in vitro, cellular or in vivo     assay (which will usually depend on the target or antigen involved),     by at least 1%, preferably at least 5%, such as at least 10% or at     least 25%, for example by at least 50%, at least 60%, at least 70%,     at least 80%, or 90% or more, compared to activity of the target or     antigen in the same assay under the same conditions but without the     presence of the construct of the invention. -    As will be clear to the skilled person, “modulating” may also     involve effecting a change (which may either be an increase or a     decrease) in affinity, avidity, specificity and/or selectivity of a     target or antigen for one or more of its ligands, binding partners,     partners for association into a homomultimeric or heteromultimeric     form, or substrates; and/or effecting a change (which may either be     an increase or a decrease) in the sensitivity of the target or     antigen for one or more conditions in the medium or surroundings in     which the target or antigen is present (such as pH, ion strength,     the presence of co-factors, etc.), compared to the same conditions     but without the presence of the construct of the invention. As will     be clear to the skilled person, this may again be determined in any     suitable manner and/or using any suitable assay known per se,     depending on the target or antigen involved. -    “Modulating” may also mean effecting a change (i.e. an activity as     an agonist, as an antagonist or as a reverse agonist, respectively,     depending on the target or antigen and the desired biological or     physiological effect) with respect to one or more biological or     physiological mechanisms, effects, responses, functions, pathways or     activities in which the target or antigen (or in which its     substrate(s), ligand(s) or pathway(s) are involved, such as its     signalling pathway or metabolic pathway and their associated     biological or physiological effects) is involved. Again, as will be     clear to the skilled person, such an action as an agonist or an     antagonist may be determined in any suitable manner and/or using any     suitable (in vitro and usually cellular or in assay) assay known per     se, depending on the target or antigen involved. In particular, an     action as an agonist or antagonist may be such that an intended     biological or physiological activity is increased or decreased,     respectively, by at least 1%, preferably at least 5%, such as at     least 10% or at least 25%, for example by at least 50%, at least     60%, at least 70%, at least 80%, or 90% or more, compared to the     biological or physiological activity in the same assay under the     same conditions but without the presence of the construct of the     invention. -    Modulating may for example also involve allosteric modulation of     the target or antigen; and/or reducing or inhibiting the binding of     the target or antigen to one of its substrates or ligands and/or     competing with a natural ligand, substrate for binding to the target     or antigen. Modulating may also involve activating the target or     antigen or the mechanism or pathway in which it is involved.     Modulating may for example also involve effecting a change in     respect of the folding or confirmation of the target or antigen, or     in respect of the ability of the target or antigen to fold, to     change its confirmation (for example, upon binding of a ligand), to     associate with other (sub)units, or to disassociate. -    Modulating may for example also involve effecting a change in the     ability of the target or antigen to transport other compounds or to     serve as a channel for other compounds (such as ions). -    Modulating may be reversible or irreversible, but for     pharmaceutical and pharmacological purposes will usually be in a     reversible manner. -   q) In respect of a target or antigen, the term “interaction site” on     the target or antigen means a site, epitope, antigenic determinant,     part, domain or stretch of amino acid residues on the target or     antigen that is a site for binding to a ligand, receptor or other     binding partner, a catalytic site, a cleavage site, a site for     allosteric interaction, a site involved in multimerisation (such as     homomerization or heterodimerization) of the target or antigen; or     any other site, epitope, antigenic determinant, part, domain or     stretch of amino acid residues on the target or antigen that is     involved in a biological action or mechanism of the target or     antigen. More generally, an “interaction site” can be any site,     epitope, antigenic determinant, part, domain or stretch of amino     acid residues on the target or antigen to which an amino acid     sequence or polypeptide of the invention can bind such that the     target or antigen (and/or any pathway, interaction, signalling,     biological mechanism or biological effect in which the target or     antigen is involved) is modulated (as defined herein). -   r) An amino acid sequence or polypeptide is said to be “specific     for” a first target or antigen compared to a second target or     antigen when is binds to the first antigen with an affinity (as     described above, and suitably expressed as a K_(D) value, K_(A)     value, K_(off) rate and/or K_(on) rate) that is at least 10 times,     such as at least 100 times, and preferably at least 1000 times, and     up to 10.000 times or more better than the affinity with which said     amino acid sequence or polypeptide binds to the second target or     polypeptide. For example, the first antigen may bind to the target     or antigen with a K_(D) value that is at least 10 times less, such     as at least 100 times less, and preferably at least 1000 times less,     such as 10.000 times less or even less than that, than the K_(D)     with which said amino acid sequence or polypeptide binds to the     second target or polypeptide. Preferably, when an amino acid     sequence or polypeptide is “specific for” a first target or antigen     compared to a second target or antigen, it is directed against (as     defined herein) said first target or antigen, but not directed     against said second target or antigen. -   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are     used interchangeably herein to mean the ability of an amino acid     sequence or other binding agents (such as a Nanobody, polypeptide or     compound or construct of the invention) to interfere with the     binding of other amino acid sequences or binding agents of the     invention to a given target. The extend to which an amino acid     sequence or other binding agents of the invention is able to     interfere with the binding of another to HER2, and therefore whether     it can be said to cross-block according to the invention, can be     determined using competition binding assays. One particularly     suitable quantitative cross-blocking assay uses a Biacore instrument     which can measure the extent of interactions using surface plasmon     resonance technology. Another suitable quantitative cross-blocking     assay uses an ELISA-based approach to measure competition between     amino acid sequences or other binding agents in terms of their     binding to the target. The following generally describes a suitable     Biacore assay for determining whether an amino acid sequence or     other binding agent cross-blocks or is capable of cross-blocking     according to the invention. It will be appreciated that the assay     can be used with any of the amino acid sequences or other binding     agents described herein. The Biacore machine (for example the     Biacore 3000) is operated in line with the manufacturer's     recommendations. Thus in one cross-blocking assay, the target     protein is coupled to a CM5 Biacore chip using standard amine     coupling chemistry to generate a surface that is coated with the     target. Typically 200-800 resonance units of the target would be     coupled to the chip (an amount that gives easily measurable levels     of binding but that is readily saturable by the concentrations of     test reagent being used). Two test amino acid sequences (termed A*     and B*) to be assessed for their ability to cross-block each other     are mixed at a one to one molar ratio of binding sites in a suitable     buffer to create the test mixture. When calculating the     concentrations on a binding site basis the molecular weight of an     amino acid sequence is assumed to be the total molecular weight of     the amino acid sequence divided by the number of target binding     sites on that amino acid sequence. The concentration of each amino     acid sequence in the test mix should be high enough to readily     saturate the binding sites for that amino acid sequence on the     target molecules captured on the Biacore chip. The amino acid     sequences in the mixture are at the same molar concentration (on a     binding basis) and that concentration would typically be between     1.00 and 1.5 micromolar (on a binding site basis). Separate     solutions containing A* alone and B* alone are also prepared. A* and     B* in these solutions should be in the same buffer and at the same     concentration as in the test mix. The test mixture is passed over     the target-coated. Biacore chip and the total amount of binding     recorded. The chip is then treated in such a way as to remove the     bound amino acid sequences without damaging the chip-bound target.     Typically this is done by treating the chip with 30 mM HCl for 60     seconds. The solution of A* alone is then passed over the     target-coated surface and the amount of binding recorded. The chip     is again treated to remove all of the bound amino acid sequences     without damaging the chip-bound target. The solution of B* alone is     then passed over the target-coated surface and the amount of binding     recorded. The maximum theoretical binding of the mixture of A* and     B* is next calculated, and is the sum of the binding of each amino     acid sequence when passed over the target surface alone. If the     actual recorded binding of the mixture is less than this theoretical     maximum then the two amino acid sequences are cross-blocking each     other. Thus, in general, a cross-blocking amino acid sequence or     other binding agent according to the invention is one which will     bind to the target in the above Biacore cross-blocking assay such     that during the assay and in the presence of a second amino acid     sequence or other binding agent of the invention the recorded     binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum     theoretical binding, specifically between 75% and 0.1% (e.g. 75% to     4%) of the maximum theoretical binding, and more specifically     between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding     (as just defined above) of the two amino acid sequences or binding     agents in combination. The Biacore assay described above is a     primary assay used to determine if amino acid sequences or other     binding agents cross-block each other according to the invention. On     rare occasions particular amino acid sequences or other binding     agents may not bind to target coupled via amine chemistry to a CM5     Biacore chip (this usually occurs when the relevant binding site on     target is masked or destroyed by the coupling to the chip). In such     cases cross-blocking can be determined using a tagged version of the     target, for example a N-terminal His-tagged version. In this     particular format, an anti-His amino acid sequence would be coupled     to the Biacore chip and then the His-tagged target would be passed     over the surface of the chip and captured by the anti-His amino acid     sequence. The cross blocking analysis would be carried out     essentially as described above, except that after each chip     regeneration cycle, new His-tagged target would be loaded back onto     the anti-His amino acid sequence coated surface. In addition to the     example given using N-terminal His-tagged [target], C-terminal     His-tagged target could alternatively be used. Furthermore, various     other tags and tag binding protein combinations that are known in     the art could be used for such a cross-blocking analysis (e.g. HA     tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies;     biotin tag with streptavidin). -    The following generally describes an ELISA assay for determining     whether an amino acid sequence or other binding agent directed     against a target cross-blocks or is capable of cross-blocking as     defined herein. It will be appreciated that the assay can be used     with any of the amino acid sequences (or other binding agents such     as polypeptides of the invention) described herein. The general     principal of the assay is to have an amino acid sequence or binding     agent that is directed against the target coated onto the wells of     an ELISA plate. An excess amount of a second, potentially     cross-blocking, anti-target amino acid sequence is added in solution     (i.e. not bound to the ELISA plate). A limited amount of the target     is then added to the wells. The coated amino acid sequence and the     amino acid sequence in solution compete for binding of the limited     number of target molecules. The plate is washed to remove excess     target that has not been bound by the coated amino acid sequence and     to also remove the second, solution phase amino acid sequence as     well as any complexes formed between the second, solution phase     amino acid sequence and target. The amount of bound target is then     measured using a reagent that is appropriate to detect the target.     An amino acid sequence in solution that is able to cross-block the     coated amino acid sequence will be able to cause a decrease in the     number of target molecules that the coated amino acid sequence can     bind relative to the number of target molecules that the coated     amino acid sequence can bind in the absence of the second, solution     phase, amino acid sequence. In the instance where the first amino     acid sequence, e.g. an Ab-X, is chosen to be the immobilized amino     acid sequence, it is coated onto the wells of the ELISA plate, after     which the plates are blocked with a suitable blocking solution to     minimize non-specific binding of reagents that are subsequently     added. An excess amount of the second amino acid sequence, i.e.     Ab-Y, is then added to the ELISA plate such that the moles of Ab-Y     target binding sites per well are at least 10 fold higher than the     moles of Ab-X target binding sites that were used, per well, during     the coating of the ELISA plate. Target is then added such that the     moles of target added per well are at least 25-fold lower than the     moles of Ab-X target binding sites that were used for coating each     well. Following a suitable incubation period the ELISA plate is     washed and a reagent for detecting the target is added to measure     the amount of target specifically hound by the coated anti-target     amino acid sequence (in this case Ab-X). The background signal for     the assay is defined as the signal obtained in wells with the coated     amino acid sequence (in this case Ab-X), second solution phase amino     acid sequence (in this case Ab-Y), target] buffer only (i.e. without     target) and target detection reagents. The positive control signal     for the assay is defined as the signal obtained in wells with the     coated amino acid sequence (in this case Ab-X), second solution     phase amino acid sequence buffer only (i.e. without second solution     phase amino acid sequence), target and target detection reagents.     The ELISA assay may be run in such a manner so as to have the     positive control signal be at least 6 times the background signal.     To avoid any artefacts (e.g. significantly different affinities     between Ab-X and Ab-Y for the target) resulting from the choice of     which amino acid sequence to use as the coating amino acid sequence     and which to use as the second (competitor) amino acid sequence, the     cross-blocking assay may to be run in two formats: 1) format 1 is     where Ab-X is the amino acid sequence that is coated onto the ELISA     plate and Ab-Y is the competitor amino acid sequence that is in     solution and 2) format 2 is where Ab-Y is the amino acid sequence     that is coated onto the ELISA plate and Ab-X is the competitor amino     acid sequence that is in solution. Ab-X and Ab-Y are defined as     cross-blocking if, either in format 1 or in format 2, the solution     phase anti-target amino acid sequence is able to cause a reduction     of between 60% and 100%, specifically between 70% and 100%, and more     specifically between 80% and 100%, of the target detection signal     {i.e. the amount of target bound by the coated amino acid sequence)     as compared to the target detection signal obtained in the absence     of the solution phase anti-target amino acid sequence (i.e. the     positive control wells). -   t) An amino acid sequence is said to be “cross-reactive” for two     different antigens or antigenic determinants (such as serum albumin     from two different species of mammal, such as human serum albumin     and cyno serum albumin) if it is specific for (as defined herein)     both these different antigens or antigenic determinants. -   u) By binding that is “essentially independent of the pH” is     generally meant herein that the association constant (K_(A)) of the     amino acid sequence with respect to the serum protein (such as serum     albumin) at the pH value(s) that occur in a cell of an animal or     human body (as further described herein) is at least 5%, such as at     least 10%, preferably at least 25%, more preferably at least 50%,     even more preferably at least 60%, such as even more preferably at     least 70%, such as at least 80% or 90% or more (or even more than     100%, such as more than 110%, more than 120% or even 130% or more,     or even more than 150%, or even more than 200%) of the association     constant (K_(A)) of the amino acid sequence with respect to the same     serum protein at the pH value(s) that occur outside said cell.     Alternatively, by binding that is “essentially independent of the     pH” is generally meant herein that the k_(off) rate (measured by     Biacore) of the amino acid sequence with respect to the serum     protein (such as serum albumin) at the pH value(s) that occur in a     cell of an animal or human body (as e.g. further described herein,     e.g. pH around 5.5, e.g. 5.3 to 5.7) is at least 5%, such as at     least 10%, preferably at least 25%, more preferably at least 50%,     even more preferably at least 60%, such as even more preferably at     least 70%, such as at least 80% or 90% or more (or even more than     100%, such as more than 110%, more than 120% or even 130% or more,     or even more than 150%, or even more than 200%) of the k_(off) rate     of the amino acid sequence with respect to the same serum protein at     the pH value(s) that occur outside said cell, e.g. pH 7.2 to 7.4. By     “the pH value(s) that occur in a cell of an animal or human body” is     meant the pH value(s) that may occur inside a cell, and in     particular inside a cell that is involved in the recycling of the     serum protein. In particular, by “the pH value(s) that occur in a     cell of an animal or human body” is meant the pH value(s) that may     occur inside a (sub)cellular compartment or vesicle that is involved     in recycling of the serum protein (e.g. as a result of pinocytosis,     endocytosis, transcytosis, exocytosis and phagocytosis or a similar     mechanism of uptake or internalization into said cell), such as an     endosome, lysosome or pinosome. -   v) As further described herein, the total number of amino acid     residues in a Nanobody can be in the region of 110-120, is     preferably 112-115, and is most preferably 113. It should however be     noted that parts, fragments, analogs or derivatives (as further     described herein) of a Nanobody are not particularly limited as to     their length and/or size, as long as such parts, fragments, analogs     or derivatives meet the further requirements outlined herein and are     also preferably suitable for the purposes described herein; -   w) The amino acid residues of a Nanobody are numbered according to     the general numbering for V_(H) domains given by Kabat et al.     (“Sequence of proteins of immunological interest”, US Public Health     Services, NIH Bethesda, Md., Publication No. 91), as applied to     V_(HH) domains from Camelids in the article of Riechmann and     Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195     (see for example FIG. 2 of this publication); or referred to herein.     According to this numbering, FR1 of a Nanobody comprises the amino     acid residues at positions 1-30, CDR1 of a Nanobody comprises the     amino acid residues at positions 31-35, FR2 of a Nanobody comprises     the amino acids at positions 36-49, CDR2 of a Nanobody comprises the     amino acid residues at positions 50-65, FR3 of a Nanobody comprises     the amino acid residues at positions 66-94, CDR3 of a Nanobody     comprises the amino acid residues at positions 95-102, and FR4 of a     Nanobody comprises the amino acid residues at positions 103-113. [In     this respect, it should be noted that—as is well known in the art     for V_(H) domains and for V_(HH) domains—the total number of amino     acid residues in each of the CDR's may vary and may not correspond     to the total number of amino acid residues indicated by the Kabat     numbering (that is, one or more positions according to the Kabat     numbering may not be occupied in the actual sequence, or the actual     sequence may contain more amino acid residues than the number     allowed for by the Kabat numbering). This means that, generally, the     numbering according to Kabat may or may not correspond to the actual     numbering of the amino acid residues in the actual sequence.     Generally, however, it can be said that, according to the numbering     of Kabat and irrespective of the number of amino acid residues in     the CDR's, position I according to the Kabat numbering corresponds     to the start of FR1 and vice versa, position 36 according to the     Kabat numbering corresponds to the start of FR2 and vice versa,     position 66 according to the Kabat numbering corresponds to the     start of FR3 and vice versa, and position 103 according to the Kabat     numbering corresponds to the start of FR4 and vice versa].

Alternative methods for numbering the amino acid residues of V_(H) domains, which methods can also be applied in an analogous manner to V_(HH) domains from Camelids and to Nanobodies, are the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called “AbM definition” and the so-called “contact definition”. However, in the present description, claims and figures, the numbering according to Kabat as applied to V_(HH) domains by Riechmann and Muyldermans will be followed, unless indicated otherwise; and

-   x) The Figures, Sequence Listing and the Experimental Part/Examples     are only given to further illustrate the invention and should not be     interpreted or construed as limiting the scope of the invention     and/or of the appended claims in any way, unless explicitly     indicated otherwise herein.

For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the prior art cited herein, to the review article by Muyldermans in Reviews in Molecular Biotechnology 74(2001), 277-302; as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (=EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application WO 06/040153, which list and references are incorporated herein by reference.

In accordance with the terminology used in the art (see the above references), the variable domains present in naturally occurring heavy chain antibodies will also be referred to as “V_(HH) domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “V_(H) domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have a number of unique structural characteristics and functional properties which make isolated V_(HH) domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring V_(HH) domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, V_(HH) domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the V_(HH) domains from the V_(H) and V_(L) domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a V_(H) domain covalently linked to a V_(L) domain).

Because of these unique properties, the use of V_(HH) domains and Nanobodies as single antigen-binding proteins or as antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional V_(H) and V_(L), domains, scFv's or conventional antibody fragments (such as Fab- or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high         affinity and with high selectivity, so that there is no need to         have two separate domains present, nor to assure that these two         domains are present in the right spatial conformation and         configuration (i.e. through the use of especially designed         linkers, as with scFv's);     -   V_(HH) domains and Nanobodies can be expressed from a single         gene and require no post-translational folding or modifications;     -   V_(HH) domains and Nanobodies can easily be engineered into         multivalent and multispecific formats (as further discussed         herein);     -   V_(HH) domains and Nanobodies are highly soluble and do not have         a tendency to aggregate (as with the mouse-derived “dAb's”         described by Ward et al., Nature, Vol. 341, 1989, p. 544);     -   V_(HH) domains and Nanobodies are highly stable to heat, pH,         proteases and other denaturing agents or conditions (see for         example Ewert et al, supra);     -   V_(HH) domains and Nanobodies are easy and relatively cheap to         prepare, even on a scale required for production. For example,         V_(HH) domains, Nanobodies and proteins/polypeptides containing         the same can be produced using microbial fermentation (e.g. as         further described below) and do not require the use of mammalian         expression systems, as with for example conventional antibody         fragments;     -   V_(HH) domains and Nanobodies are relatively small         (approximately 15 kDa, or 10 times smaller than a conventional         IgG) compared to conventional 4-chain antibodies and         antigen-binding fragments thereof, and therefore show high(er)         penetration into tissues (including but not limited to solid         tumors and other dense tissues) than such conventional 4-chain         antibodies and antigen-binding fragments thereof; V_(HH) domains         and Nanobodies can show so-called cavity-binding properties         (inter alia due to their extended CDR3 loop, compared to         conventional V_(H) domains) and can therefore also access         targets and epitopes not accessible to conventional 4-chain         antibodies and antigen-binding fragments thereof. For example,         it has been shown that V_(HH) domains and Nanobodies can inhibit         enzymes (see for example WO 97/49805; Transue et al., Proteins         1998 Sep. 1; 32(4): 515-22; Lauwereys et al., EMBO J. 1998 Jul.         1; 17(13): 3512-20).

In a specific and preferred aspect, the invention provides Nanobodies against HER2, and in particular Nanobodies against HER2 from a warm-blooded animal, and more in particular Nanobodies against HER2 from a mammal, and especially Nanobodies against human HER2; as well as proteins and/or polypeptides comprising at least one such Nanobody.

In particular, the invention provides Nanobodies against HER2, and proteins and/or polypeptides comprising the same, that have improved therapeutic and/or pharmacological properties and/or other advantageous properties (such as, for example, improved ease of preparation and/or reduced costs of goods), compared to conventional antibodies against HER2 or fragments thereof, compared to constructs that could be based on such conventional antibodies or antibody fragments (such as Fab′ fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and other multispecific constructs (see for example the review by Holliger and Hudson, Nat. Biotechnol. 2005 September; 23(9):1126-36)), and also compared to the so-called “dAb's” or similar (single) domain antibodies that may be derived from variable domains of conventional antibodies. These improved and advantageous properties will become clear from the further description herein, and for example include, without limitation, one or more of:

-   -   increased affinity and/or avidity for HER2, either in a         monovalent format, in a multivalent format (for example in a         bivalent format) and/or in a multispecific format (for example         one of the multispecific formats described hereinbelow);     -   better suitability for formatting in a multivalent format (for         example in a bivalent format);     -   better suitability for formatting in a multispecific format (for         example one of the multispecific formats described hereinbelow);     -   improved suitability or susceptibility for “humanizing”         substitutions (as defined herein);     -   less immunogenicity, either in a monovalent format, in a         multivalent format (for example in a bivalent format) and/or in         a multispecific format (for example one of the multispecific         formats described hereinbelow);     -   increased stability, either in a monovalent format, in a         multivalent format (for example in a bivalent format) and/or in         a multispecific format (for example one of the multispecific         formats described hereinbelow);     -   increased specificity towards HER2, either in a monovalent         format, in a multivalent format (for example in a bivalent         format) and/or in a multispecific format (for example one of the         multispecific formats described hereinbelow);     -   decreased or where desired increased cross-reactivity with HER2         from different species;         and/or     -   one or more other improved properties desirable for         pharmaceutical use (including prophylactic use and/or         therapeutic use) and/or for diagnostic use (including but not         limited to use for imaging purposes), either in a monovalent         format, in a multivalent format (for example in a bivalent         format) and/or in a multispecific format (for example one of the         multispecific formats described hereinbelow).

As generally described herein for the amino acid sequences of the invention, the Nanobodies of the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more Nanobodies of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other antigenic determinants on HER2 and/or against one or more other targets than HER2), so as to provide a monovalent, multivalent, multiparatopic or multispecific polypeptide of the invention, respectively, all as described herein. In particular, such a protein or polypeptide may comprise or essentially consist of one or more Nanobodies of the invention and optionally one or more (other) Nanobodies (i.e. directed against one or more other antigenic determinants on HER2 and/or against other targets than HER2), all optionally linked via one or more suitable linkers, so as to provide a monovalent, multivalent, multiparatopic or multispecific Nanobody construct, respectively, as further described herein. Such proteins or polypeptides may also be in essentially isolated form (as defined herein).

In a Nanobody of the invention, the binding site for binding against HER2 is preferably formed by the CDR sequences. Optionally, a Nanobody of the invention may also, and in addition to the at least one binding site for binding against HER2, contain one or more further binding sites for binding against other antigens, proteins or targets. For methods and positions for introducing such second binding sites, reference is for example made to Keck and Huston, Biophysical Journal, 71, October 1996, 2002-2011; EP 0 640 130; and WO 06/07260.

As generally described herein for the amino acid sequences of the invention, when a Nanobody of the invention (or a polypeptide of the invention comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably directed against human HER2; whereas for veterinary purposes, it is preferably directed against HER2 from the species to be treated.

Also, as with the amino acid sequences of the invention, a Nanobody of the invention may or may not be cross-reactive (i.e. directed against HER2 from two or more species of mammal, such as against human HER2 and HER2 from at least one of the species of mammal mentioned herein).

Also, again as generally described herein for the amino acid sequences of the invention, the Nanobodies of the invention may generally be directed against any antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of HER2 However, it is generally assumed and preferred that the Nanobodies of the invention (and polypeptides comprising the same) are directed against the Herceptin® binding site on HER2 or the Omnitarg binding site on HER2.

As already described herein, the amino acid sequence and structure of a Nanobody can be considered—without however being limited thereto—to be comprised of four framework regions or “FR's” (or sometimes also referred to as “FW's”), which are referred to in the art and herein as “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”, respectively; which framework regions are interrupted by three complementary determining regions or “CDR's”, which are referred to in the art as “Complementarity Determining Region 1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3”, respectively. Some preferred framework sequences and CDR's (and combinations thereof) that are present in the Nanobodies of the invention are as described herein. Other suitable CDR sequences can be obtained by the methods described herein.

According to a non-limiting but preferred aspect of the invention, (the CDR sequences present in) the Nanobodies of the invention are such that:

-   -   the Nanobodies can bind to HER2 with a dissociation constant         (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably         10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to         10⁻¹² moles/liter (i.e. with an association constant (K_(A)) of         10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²         liter/moles or more and more preferably 10⁸ to 10¹²         liter/moles);         and/or such that:     -   the Nanobodies can bind to HER2 with a k_(on)-rate of between         10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹         and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹         s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;         and/or such that they:     -   the Nanobodies can bind to HER2 with a k_(off) rate between 1         s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible         complex with a t_(1/2) of multiple days), preferably between         10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶         s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, (the CDR sequences present in) the Nanobodies of the invention are such that: a monovalent Nanobody of the invention (or a polypeptide that contains only one Nanobody of the invention) is preferably such that it will bind to HER2 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

The affinity of the Nanobody of the invention against HER2 can be determined in a manner known per se, for example using the general techniques for measuring K_(D). K_(A), k_(off) or k_(on) mentioned herein, as well as some of the specific assays described herein.

Some preferred IC 50 values for binding of the Nanobodies of the invention (and of polypeptides comprising the same) to HER2 will become clear from the further description and examples herein.

In a preferred but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against HER2, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     401-675;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     401-675;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 951-1225;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775;     or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against HER2, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     401-675;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     401-675;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 951-1225;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     951-1225;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's:     1501-1775;     or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of the invention, when a Nanobody of the invention contains one or more CDR1 sequences according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)     and/or c) is preferably, and compared to the corresponding CDR     according to a), a conservative amino acid substitution (as defined     herein);     and/or -   ii) the CDR according to b) and/or c) preferably only contains amino     acid substitutions, and no amino acid deletions or insertions,     compared to the corresponding CDR according to a);     and/or -   iii) the CDR according to b) and/or c) may be a CDR that is derived     from a CDR according to a) by means of affinity maturation using one     or more techniques of affinity maturation known per se.

Similarly, when a Nanobody of the invention contains one or more CDR2 sequences according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)     and/or 1) is preferably, and compared to the corresponding CDR     according to d), a conservative amino acid substitution (as defined     herein);     and/or -   ii) the CDR according to e) and/or f) preferably only contains amino     acid substitutions, and no amino acid deletions or insertions,     compared to the corresponding CDR according to d);     and/or -   iii) the CDR according to e) and/or f) may be a CDR that is derived     from a CDR according to d) by means of affinity maturation using one     or more techniques of affinity maturation known per se.

Also, similarly, when a Nanobody of the invention contains one or more CDR3 sequences according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)     and/or i) is preferably, and compared to the corresponding CDR     according to g), a conservative amino acid substitution (as defined     herein);     and/or -   ii) the CDR according to h) and/or i) preferably only contains amino     acid substitutions, and no amino acid deletions or insertions,     compared to the corresponding CDR according to g);     and/or -   iii) the CDR according to h) and/or i) may be a CDR that is derived     from a CDR according to g) by means of affinity maturation using one     or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally apply to any Nanobody of the invention that comprises one or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e), f), h) or i), respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more of the CDR's explicitly listed above are particularly preferred; Nanobodies comprising two or more of the CDR's explicitly listed above are more particularly preferred; and Nanobodies comprising three of the CDR's explicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDR sequences, as well as preferred combinations of CDR sequences and framework sequences, are mentioned in Table A-1 below, which lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) Nanobodies of the invention. As will be clear to the skilled person, a combination of CDR1, CDR2 and CDR3 sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the same line in Table A-1) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences mentioned in Table A-1). Also, a combination of CDR sequences and framework sequences that occur in the same clone (i.e. CDR sequences and framework sequences that are mentioned on the same line in Table A-1) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences and framework sequences mentioned in Table A-1, as well as combinations of such CDR sequences and other suitable framework sequences, e.g. as further described herein).

Also, in the Nanobodies of the invention that comprise the combinations of CDR's mentioned in Table A-1, each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and     compared to the corresponding CDR sequence mentioned in Table A-1, a     conservative amino acid substitution (as defined herein);     and/or -   ii) any such CDR sequence preferably only contains amino acid     substitutions, and no amino acid deletions or insertions, compared     to the corresponding CDR sequence mentioned in Table A-1;     and/or -   iii) any such CDR sequence is a CDR that is derived by means of a     technique for affinity maturation known per se, and in particular     starting from the corresponding CDR sequence mentioned in Table A-1.

However, as will be clear to the skilled person, the (combinations of) CDR sequences, as well as (the combinations of) CDR sequences and framework sequences mentioned in Table A-1 will generally be preferred.

TABLE A-1  Preferred combinations of CDR sequences, preferred combinations of framework sequences, and preferred combinations of framework and CDR sequences. (“ID” refers to the SEQ ID NO in the attached sequence listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID 13D11 126 EVQLVESGGGLV 401 DYGMT 676 WVRRAPGK 951 SINWSGTHTDY 1226 HPGGSLRLSCVG GLEWVS ADSVKG SGFSLD 2B4 127 EVQLVESGGGLV 402 DYAMT 677 WVRQAPGK 952 SINWSGTHTDY 1227 QPGGSLRLSCVG GLEWVS ADSVKG SGFSLD 2G2 128 EVQLVESGGGLV 403 DYGMT 678 WVRQAPGK 953 SINWSGTHTDY 1228 QPGGSLRLSCVA GLEWVS TDPVKG SGFSLD 13D2 129 EVQLVESGGGLV 404 DYGMT 679 WVRQAPGK 954 SINWSGTHTDY 1229 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2D5 130 EVQLVESGGGLV 405 DYGMT 680 WVRQAPGK 955 SINWSGTHTDY 1230 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2F4 131 EVQLVESGGGLV 406 DYGMT 681 WVRQAPGK 956 SINWSGTHTDY 1231 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2C3 132 EVQLVESGGGLV 407 DYGMT 682 WVRQAPGK 957 SINWSGTHTDY 1232 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 17E3 133 EVQLVESGGGLV 408 RYTMG 683 WYRQAPGK 958 SIDSSGGTNYA 1233 QAGGSLRLSCVA QRDLVA DSVKG SKMTFM 17H3 134 EVQLMESGGGLV 409 DYGMT 684 WVRQAPGK 959 SINWSGTHTDY 1234 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 17D2 135 EVQLVESGGGLV 410 DYGMT 685 WVRQAPGK 960 SINWSGTHTDY 1235 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2F1 136 EVQLVESGGGLV 411 DYGMT 686 WVRQAPGK 961 SINWSGTHTDY 1236 QPGGSLRLSCVA ELEWIS ADSVKG SGFSLD 2E2 137 EVQLVESGGGLV 412 DYGMT 687 WVRQAPGK 962 SINWSGTHTDY 1237 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2C2 138 EVQLVESGGGLV 413 DYAMT 688 WVRQAPGK 963 SINWSGTHTDY 1238 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2E3 139 EVQLVESGGGLV 414 DYGMT 689 WVRQAPGK 964 SINWSGTHTDY 1239 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 13B10 140 EVQLVESGGGLV 415 DYGMT 690 WVRQAPGK 965 SINWSGTHTDY 1240 QPGGSLRLSCVA GFEWVS ADSVKG SGFSLD 2D1 141 EVQLVESGGGLV 416 DYGMT 691 WVRQAPGK 966 SINWSGTHTDY 1241 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2H3 142 EVQLVESGGGLV 417 DYGMT 692 WVRQAPGK 967 SINWSGTHTDY 1242 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2H1 143 EVQLVESGGGLV 418 DYGMT 693 WVRQAPGK 968 SINWSGTHTDY 1243 QPGGSLRLSCVA GLEWVS ADSVRG SGFSLD 2C1 144 EVQLVESGGGLV 419 DYGMT 694 WVRQAPGK 969 SINWSGTHTDY 1244 QPGGSLRLSCVA GLEWVS TDSVKG SGFSLD 15C5 145 EVQLVESGGGLV 420 DYGMT 695 WVRQAPGK 970 SINWNVTHTDY 1245 QPGGSLKLSCVA GLEWVS AYSVKG SGFSLD 2B3 146 EVQLVESGGGLV 421 DYGMT 696 WVRQAPGK 971 SINWSGTHTDC 1246 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 29H2 147 EVQLVESGGGLV 422 DYGMT 697 WVRQAPGK 972 SINWSGTHTDY 1247 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 17E4 148 EVQLVESGGGLV 423 DYGMT 698 WVRQAPGK 973 SINWSGTHTDY 1248 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 17A2 149 EVQLVESGGGLV 424 DYAMT 699 WVRQAPGK 974 SINWSGTHTDY 1249 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 15D1 150 EVQLVESGGGLV 425 DYAMT 700 WVRQAPGK 975 SINWSGTHTDY 1250 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 17B8 151 EVQLVESGGGLV 426 DYGMT 701 WVRQAPGK 976 SINWSGTHTDY 1251 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 15C11 152 EVQLVESGGGLV 427 DYGMT 702 WVRQAPGK 977 SINWSGTHTDY 1252 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 15G8 153 EVQLVESGGGLV 428 DYGMT 703 WVRQAPGK 978 SINWNGTHTDY 1253 QPGGSLKLSCVA GLEWVS AYSVKG SGFSLD 17H4 154 EVQLVESGGGLV 429 NYAMT 704 WVRQAPGK 979 SINWSGTHTDY 1254 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLI 27G8 155 EVQLVESGGGLV 430 DYGMT 705 WVRQAPGK 980 SINWSGTHTDY 1255 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 38C6 156 EVQLVESGGGLV 431 DYAMT 706 WVRQAPGK 981 SINWSGTHTDY 1256 QPGGSLRLSCVG GLEWVS ADSVKG SGFSLD 2A4 157 EVQLVESGGGLV 432 DYAMS 707 WVRQAPGK 982 AINWSGSHRN 1257 QPGGSLRLSCAA GLEWVS YADSVKG SGFIFD 15G7 158 EVQLVESGGGLV 433 DYAMS 708 WVRQAPGK 983 AINWSGTHRN 1258 QPGGSLRLSCAA GLEWVS YADSVKG SGFIFD 15B7 159 EVQLVESGGGLV 434 DYAMS 709 WVRQAPGK 984 AINWSGSHRN 1259 QPGGSLKLSCAA GLEWVS YADSVKG SGFIFD 5G4 160 EVQLVESGGGLV 435 DYAMS 710 WVRQAPGK 985 SINWSGSHRN 1260 QPGGSLTLSCAG GLEWVS YADSVKG SGFIFD 13B2 161 EVQLVESGGSLV 436 DYAMS 711 WVRQAPGK 986 SINWSGTHKDY 1261 QPGGSLRLSCAA GLEWIS ADSVKG SGFTFD 2E5 162 EVQLVESGGSLV 437 DYAMS 712 WVRQAPGK 987 SINWSGTHTDY 1262 QPGESLRLSCAA GLEWIS ADSVKG SGFTFD 15G1 163 EVQLVESGGSLV 438 DYAMS 713 WVRQAPGK 988 SINWSGTHTDY 1263 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 27B1 164 EVQLVESGGSLV 439 DYAMS 714 WVRQAPGK 989 SINWSGTHTDY 1264 QPGGSLRLSCAA GLEWIS ADSVKG SGFTFD 17E7 165 EVQLVESGGSLV 440 DYAMS 715 WVRQVPGK 990 SINWSGTHTDY 1265 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 17D8 166 EVQLVESGGSLV 441 DYAMS 716 WVRQAPGK 991 SINWSGTHTDY 1266 PPGGSLRLSCAV GLEWVS TDSVKG SGFTFD 5F8 167 EVQLVESGGSLV 442 DYALS 717 WVRQAPGK 992 SINWSGTHTDY 1267 QPGGSLRLSCAA GLEWIS ADSVKG SGFTFD 2D4 168 EVQLVESGGSLV 443 DYAMT 718 WVRQAPGK 993 SINWSGTHTDY 1268 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 13D8 169 EVQLVESGGSLV 444 DYAMT 719 WVRQASGK 994 SINWSGTHTDY 1269 QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 17G8 170 EVQLVESGGSLV 445 DYAMS 720 WVRQAPGK 995 SINWSGTHTGY 1270 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2H4 171 EVQLVESGGSLV 446 DYAMT 721 WVRQAPGK 996 SINWSGTHTDY 1271 QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2F3 172 EVQLVESGGSLV 447 DYAMT 722 WVRQAPGK 997 SINWSGTHTDY 1272 QPGGSLRLSCAA GLEWVS TGSVKG SGFTFD 2F5 173 EVQLVESGGSLV 448 DYAMS 723 WVRQAPGK 998 SINWSGTHTDY 1273 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 30E10 174 KVQLVESGGSLV 449 DYAMT 724 WVRQAPGK 999 SINWSGTHTDY 1274 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 29H1 175 EVQLVESGGSLV 450 DYAMS 725 WVRQAPGK 1000 SINWSGTHTGY 1275 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 17E2 176 EVQLVESGGSLV 451 DYGMS 726 WVRQAPGK 1001 SINWSGTHTDY 1276 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2B1 177 EVQLVESGGSLV 452 DYAMT 727 WVRQAPGK 1002 SINWSGTHTDY 1277 QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2A5 178 EVQLVESGGGLV 453 DYAMT 728 WVRQAPGK 1003 SINWSGTHTDY 1278 QPGGSLRLSCAT GLEWVS TDSVKG SGFTFD 13C12 179 EVQLVESGGSLV 454 DYAMT 729 WVRQAPGK 1004 SINWSGTHTDY 1279 QPGGSLRLSCAT GLEWVS TDSVKG SGFTFD 17E10 180 EVQLVESGGSLV 455 DYAMT 730 WVRQAPGK 1005 SINWSGTHTDC 1280 QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 27D4 181 EVQLVESGGSLV 456 DYAMT 731 WVRQASGK 1006 SINWSGTHTDY 1281 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 15F9 182 EVQLVESGGSLV 457 DYAMT 732 WVRQAPGK 1007 SINWSGTHTDY 1282 QPGGSLRLSCAA GLEWVS TGSVKG SGFTFD 30H9 183 EVQLVESGGSLV 458 DYAMT 733 WVRQAPGK 1008 SINWSGTHTDY 1283 QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 39C1 184 EVQLVESGGSLV 459 DYGMS 734 WVRQAPGK 1009 SINWSGTHTDY 1284 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 27G2 185 EVQLVESGGSLV 460 DYAMT 735 WVRQTPGK 1010 SINWSGTHTDY 1285 QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2D3 186 EVQLVESGGSLV 461 DYAMS 736 WVRQVPGK 1011 SINWSGTHTDY 1286 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 5F7 187 EVQLVESGGGLV 462 INTMG 737 WYRQAPGK 1012 LISSIGDTYYAD 1287 QAGGSLRLSCAA QRELVA SVKG SGITFS 118N121_A1_4_OK/ 188 EVQLVESGGGFV 463 EYAAA 738 WFRQSPGK 1013 GIMWDGRSLF 1288 1-127 QTGGSPRLSCAA ERDLVA YADSVKG SGRSFS 47D5 189 KVQLVESGGGLV 464 FNDMA 739 WYRQAPGK 1014 LISRVGVTSSA 1289 QPGGSLRLSCAA QRELVA DSVKG SGSIFG 14B11 190 EVQLVESGGGLV 465 SYGMG 740 WFRQVPGK 1015 TINWSGVTAYA 1290 QAGGSLRLSCAA EREFVA DSVKG SGSTFS 14B10 191 EVQLVESGGGLV 466 SYGMG 741 WFRQAPGK 1016 TINWSGVTAYA 1291 QAGGSLRLSCAV EREFVA DSIKG NSRTFS 14B4 192 EVQLVESGGGLV 467 SYGMG 742 WFRQAPGK 1017 TINWSGVTAYA 1292 QAGGSLRLSCAV DREFVA DSIKG SSRAFS 14C11 193 EVQLVESGGGLV 468 SYGMG 743 WFRQAPGK 1018 TINWSGATAYA 1293 QAGGSLRLSCAV EREFVA DSIKG NSRTFS 14B5 194 EVQLVESGGGLV 469 SYGMG 744 WFRQAPGK 1019 TINWSGVTAYA 1294 QAGGSLRLSCAV DREFVA DSIKG SSRAFS 14C6 195 EVQLVESGGGSV 470 SYGMG 745 WFRQAPGK 1020 TINWSGVTAYA 1295 QAGGSLRLSCVA ERAFVA DSVKG SEGTFS 14A4 196 EVQLVESGGGSV 471 SYGMG 746 WFRQAPGK 1021 TINWSGVNAYA 1296 QAGSSLTLSCVAS ERAFVA DSVKG EGTFS 14B3 197 EVQLVESGGGLV 472 SYGMG 747 WFRQAPGK 1022 TINWSGVNAYA 1297 QPGGSLTLSCVA ERAFVA DSVKG SEGTFS 14C1 198 EVQLVESGGGSV 473 SYGMG 748 WFRQAPGK 1023 TINWSGVTAYA 1298 QAGGSLRLSCAA ERAFVA DSVKG SGSTFS 14A12 199 EVQLVKSGGGLV 474 SYGMG 749 WFRQAPGK 1024 TINWSGVTAYA 1299 QAGGSLRLSCAA EREFVA DSVKG SERTFS 14A2 200 EVQLVESGGGLV 475 SYGMG 750 WFRQAPGK 1025 TINWSGVTAYA 1300 QAGGSLRLSCAA EREFVA DSVKG SERTFS 14A1 201 EVQLVESGGGSV 476 SYGMG 751 WFRQAPGK 1026 TINWSGVTAYA 1301 QAGGSLRLSCAA EREFVA DSVKG SERTFS 17C3 202 EVQLVESGGGLV 477 RYDMG 752 WYRQAPGQ 1027 AISGAGDINYA 1302 QAGGSLRLSCAA QREWVA DSVKG NGLTFR 46D3 203 KVQLVESGGGLV 478 EYSMG 753 WFRQAPGK 1028 TISWNYGYTYY 1303 QAGGSLRLSCAA EREFVA SDSVKG SGRTFT 27H5 204 EVQLVESGGGLV 479 DYGIG 754 WFRQASGK 1029 CITSSDGSTYY 1304 QAGGSLRLSCAA EREGVS ADSVKG SGFTFD 17C2 205 EVQLVESGGGLV 480 SYAMS 755 WVRQAPGK 1030 AVDSGGGRTD 1305 QPGGSLRLSCAA GLEWVS YAHSVKG SGFAFS 17D11 206 EVQLVESGGGLV 481 TSAMG 756 WFRQAPGK 1031 TISRGGSATYY 1306 QAGGSLRLSCTA EREFVA ADSLKG SGRTSS 15A6 207 EVQLVESGGGLV 482 TRTMA 757 WYRQAPGK 1032 TISSHGLPVYA 1307 QAGGSLRLSCVT QRDWVA DSVKG SRRPAS 17B6 208 EVQLVESGGGLV 483 TRTMA 758 WYRQAPGK 1033 TIGTSGPPRYA 1308 QPGGSLRLSCAA QRDWVA DSVKG SRIPFS 17C5 209 EVQLVESGGGLV 484 TRTMA 759 WYRQAPGK 1034 TISSHGLPVYA 1309 QAGGSLRLSCVT QRDWVA DSVKG SRRPAS 15E11 210 EVQLVESGGGLV 485 SRTMA 760 WYRQAPGK 1035 TISARGMPAYE 1310 QAGGSLRLSCVA QRDWVA DSVKG SRIPFS 15C2 211 EVQLVESGGGLV 486 TRTMA 761 WYRQAQGK 1036 TISSHGLPVYA 1311 QAGGSLRLSCVT QRDWVA DSVKG SRRPAS 2A3 212 EVQLVESGGGLV 487 TRTMA 762 WYRQAPGK 1037 TIRNGAPVYAD 1312 QAGGSLNLSCVA PRDWVA SVKG SGIPFS 27A5 213 EVQLVESGGGLV 488 TRTMA 763 WYRQPPGN 1038 TIRSGAPVYAD 1313 QAGGSLNLSCVA ERDWVA SVKG SGIPFS 2C5 214 EVQLVESGGGLV 489 TRTMA 764 WYRQTPGK 1039 TIRSGTPVYAD 1314 QAGGSLNLSCVA SRDWVA SVKG SGIPFS 27G5 215 EVQLVESGGGLV 490 IRTMA 765 WYRQTPGN 1040 TIGSSGTPAYA 1315 QPGGSLRLSCVA QRDWLA DSVKG SRIPAS 13A9 216 EVQLVESGGGLV 491 IRTMA 766 WYRQAPGK 1041 TIGTGGTPAYA 1316 QAGGSLRLSCVA QRDWVA DSFKG SRIPAS 29E9 217 EVQLVESGGGLV 492 IRTMA 767 WYRQTPGN 1042 TIGSSGTPAYA 1317 QPGGSLRLSCVA QRDWLA DSVKG SRIPAS 15D8 218 EVQLVESGGGLV 493 IRTMA 768 WYRQTPGN 1043 TIGSSGTPAYA 1318 QPGGSLKLSCVA QRDWLA DSVKG STIPAS 15G4 219 EVQLVESGGGLV 494 SRTMA 769 WYRQAPGK 1044 TIGTHGTPLYA 1319 QAGGSLRLSCVA TRDWVA DSVKG SGIPFR 15D12 220 EVQLVESGGGLV 495 RYVMG 770 WYRQGPGK 1045 TVNDGGTTSY 1320 QAGESLRLSCATS QRELVA ADSVKG GITFK 15E12 221 EVQLMESGGGLV 496 RYDMG 771 WYRQAPGQ 1046 AISGAGDINYA 1321 QAGGSLRLSCAA QREWVA DSVKG NGLTFR 13D7 222 EVQLVESGGGLV 497 RYDMG 772 WYRQAPGQ 1047 AISGAGDINYA 1322 QAGGSLRLSCAA QREWVA DSVKG NGLTFR 13A8 223 EVQLVESGGGLV 498 FSRRTMA 773 WYRQAPGK 1048 TIAGDGSTVYA 1323 QPGGSLRLSCAA QRDWVA DSMKG SGLGIA 15A4 224 EVQLVESGGGLV 499 FSRRTMA 774 WYRQAPGK 1049 TIAGDGSTVYA 1324 QPGGSLRLSCAA QRDWVA DSMKG SGLGIA 17F7 225 EVQLVESGGGLV 500 IRVMA 775 WYRQPPGK 1050 TISSDGTANYA 1325 QAGGSLRLSCVA QRDWVG DSVKG SGIAQS 15C8 226 EVQLVESGGGLV 501 IRTMA 776 WYRQAPGK 1051 TSDSGGTTLYA 1326 QAGGSLRLSCAA QRDWVA DSVKG SGIAFR 17A10 227 EVQLVESGGGLV 502 RAIA 777 WYRQAPGK 1052 TSGTGYGATY 1327 QAGGSLRLSCVA QRDWVA DDSVKG SGIPSI 27D3 228 EVQLMESGGGLV 503 FSRRTMA 778 WYRQAPGK 1053 TIAGDGSTVYA 1328 QPGGSLRLSCAA QRDWVA DSMKG SGLGIA 13B12 229 EVQLVESGGGLV 504 IRTMA 779 WYRQAPGK 1054 TIGSDGTTJYAD 1329 QAGGSLRLSCAA QRDWVA SVKG SGIAFR 15B2 230 EVQLVESGGGLV 505 IRAMA 780 WYRQAPGR 1055 TIYSPSGSAVY 1330 QAGGSLRLSCVV QRDWVA ADSVKG SGIPSS 15B11 231 EVQLVESGGGSV 506 IRAMA 781 WYRQAPGR 1056 TIYSRSGGAVY 1331 QAGGSLRLSCVV QRDWVA ADSVKG SGIPSS 13C9 232 EVQLVESGGGLV 507 HAMA 782 WYRQAPGK 1057 TTYSRGGTTYN 1332 QAGGSLRLSCVA QRDWGA DSAKG SGIPSI 17D5 233 EVQLVESGGGLV 508 IRTMA 783 WYRQAPGK 1058 SIGTRGAPVYA 1333 QPGGSLRLSCAA QRDWVA DSVNG SGIIGT 27B5 234 EVQLVESGGGLV 509 IRTMA 784 WYRQAPGK 1059 TSDSGGTTLYA 1334 QAGGSLRLPCAA QRDWVA DSVKG SGIAFR 27C7 235 EVQLVESGGGLV 510 IRTMA 785 WYRQAPGK 1060 TSDSGGTTLYA 1335 QAGGSLRLSCAA QRDWVA DSVKG SGIAFR 13D4 236 EVQLVESGGGLV 511 IRAMA 786 WYRQAPGR 1061 TIYSPSGSAVY 1336 QAGGSLRLSCVV QRDWVA ADSVKG SGIPSS 15G5 237 EVQLVESGGGLV 512 IRAMA 787 WYRQAPGR 1062 TIYSPSGSAVY 1337 QAGGSLRLSCVV QRDWVA ADSVKG SGIPST 13C4 238 EVQLVESGGGLV 513 IRAMA 788 WYRQAPGR 1063 TIYSPSGSAVY 1338 QAGGSLRLSCVV QRDWVA ADSVKG SGIPSS 46G1 239 EVQLVESGGGLV 514 DDAMG 789 WFRQAPGK 1064 SLYLNGDYPYY 1339 QAGGSLRLSCAA ERECVA ADSVKG SGRTFS 46E4 240 EVQLVESGGGLV 515 DDAVG 790 WFRQAPGK 1065 SMYLDGDYPY 1340 QAGGSLRLSCAA ERECVA YADSVKG SGRAFK 17B5 241 EVQLVESGGGLV 516 TDMMG 791 WYRQAPGK 1066 SITKFGSTNYA 1341 QTGGSLRLSCAA QREFVA DSVKG SGSTFR 15C9 242 EVQLVESGGGLV 517 LRAMA 792 WYRQAPGR 1067 TSSNTGGTTYD 1342 QAGGSLKLSCVN QRDWVA DSVKG SGIPST 13D10 243 EVQLVESGGGLV 518 DSNAIG 793 WFRQAPGK 1068 CIASSDGSTYY 1343 QPGGSLRLSCAA EREEVS AESVKG SSVITL 17C6 244 EVQLVESGGGLV 519 LDIMA 794 WYRQAPEK 1069 SVSGGGNSDY 1344 QAGGSLTLSCAA QRELVA ASSVKG SGSTSS 15A2 245 EVQLVESGGGLA 520 TRVMA 795 WYRQTPGK 1070 SMRGSGSTNY 1345 QAGGSLSLSCAA QREFVA ADSARG SGRFFS 17A8 246 EVQLVESGGGLV 521 TRVMA 796 WYRQTPGK 1071 SMRGSGSTNY 1346 QAGGSLSLSCAA QREFVA ADSVRG SGRFFS 15G10 247 EVQLVESGGGLV 522 TRVMA 797 WYRQTPGK 1072 SMRGSGSTNY 1347 QAGGSLSLSCAA QREFVA ADSARG SGRFFS 27A3 248 EVQLVESGGGLV 523 TRVMA 796 WYRQTPGK 1073 SMRGSGSTNY 1348 QAGGSLSLSCVA QREFVA ADSVRG SGRFFS 17H10 249 EVQLVESGGGLV 524 TRVMA 799 WYRQTPGN 1074 TIHSSGSTIYAD 1349 QAGGSLSLSCSA QREFVA SVRG SGRFFS 30D10 250 EVQLVESGGGLV 525 IRTMA 800 WYRQPPGN 1075 TIGSNGFATYP 1350 QAGGSLTLSCTAS QREWVA DSVKG ETTVR 15H4 251 EVQLVESGGGLV 526 FNTVA 801 WYRQAPGE 1076 TISRQGMSTYP 1351 QAGGSLTLSCAP QREWVA DSVKG SESTVS 17B7 252 EVQLVESGGGLV 527 FRTMA 802 WYRQAPGK 1077 TIGSDGLANYA 1352 QAGGSLRLSCAA QREWVA DSVKG SGIISS 15D2 253 EVQLVESGGGLV 528 IRAMA 803 WYRQAPGK 1078 TIGSSGHPVYT 1353 QAGGSLRLSCVV QRDWVA DSVKG SGVFGP 17G5 254 EVQLVESGGGLV 529 FSSRTMA 804 WYRQAPGK 1079 TIGSGGTTNYA 1354 QPGGSLRLSCAA QRDWVA DSVKG SGIGIA 15B6 255 EVQLVESGGGLV 530 FRTMA 805 WYRQAPGN 1080 TIGSAGLASYA 1355 QPGGSLRLSCAA QRDWVA DSVRG SGIIGS 27F2 256 EVQLVESGGGLV 531 FRTLA 806 WYRQAPGK 1081 TISSAGGTAYA 1356 QAGGSLRLSCAA QRDWVA DAVKG SGIISS 17F5 257 EVQLVESGGGLV 532 FSRRTMA 807 WYRQAPGK 1082 TIAGDGSTVYA 1357 QPGGSLRLSCAA QRDWVA DSMKG SGLGIA 17B2 258 EVQLVESGGGLV 533 NYAMT 808 WVRQAPGK 1083 GVGGDGVGSY 1358 QPGGSLRLSCAG GLEWVS ADSVKG SGFTFS 27H4 259 EVQLVESGGGLV 534 RYTMG 809 WYRQAPGK 1084 SIDASGGTNYA 1359 QAGGSLRLSCVA QRDLVA DSVKG SKMTFM 13A4 260 EVQLVESGGGLV 535 RYTMG 810 WYRQAPGK 1085 SIDSSGGTNYA 1360 QAGGSLRLSCVA QRDLVA DSVKG SKMTFM 2A1 261 EVQLVESGGGLV 536 RYIMD 811 WYRQAPGK 1086 SINSDGSTGYT 1361 QAGGSLRLSCVA QRELVA DSVKG SKITFR 15E10 262 EVQLVESGGGLV 537 RYTMG 812 WYRQAPGK 1087 EISSADEPSFA 1362 QAGGSLKLSCVA ERELVA DAVKG SGITFF 27E7 263 EVQLVESGGGLV 538 RYDMG 813 WYRQFPGK 1088 TILSEGDTNYV 1363 QAGGSLRLSCAA ERELVA DPVKG SGITFR 47E5 264 EVQLVESGGGLV 539 FDSMG 814 WYRQAPGN 1089 IISNGGTTSYR 1364 QAGGSLRLSCAA ERILVA DSVKG SASIFG 2G4 265 EVQLVESGGGLV 540 HNAMG 815 WYRQAPGK 1090 YITINGIANYVD 1365 QAGGSLRLSCAA QRELVT SVKG SGNIFS 14D4 266 EVQLVESGGGLV 541 TYVMG 816 WFRQAPGD 1091 HIFRSGITSYAS 1366 QAGDSLRLSCAA GREFVA SVKG SGRALD 17A5 267 EVQLVESGGGLV 542 DYSMS 817 WVRQATGK 1092 GISWNGGSTN 1367 QPGGSLRLSCAA GLEWVS YADSVKG SGFTFD 15D10 268 EVQLVESGGGLV 543 SYRMY 818 WVRQAPGK 1093 AIKPDGSITYYA 1368 QPGGSLKLSCAA GLEWVS DSVKG SGFTFS 13C2 269 EVQLVESGGGLV 544 INRMA 819 WYRQSPGK 1094 AVDNDDNTEY 1369 QAGGSLRLSCAA QRELVA SDSVAG SGSTFS 17G11 270 EVQLVESGGGLV 545 INRWG 820 WYRQAPGK 1095 AIDDGGNTEYS 1370 QAGGSLRLSCAA QRELVA DFVNG SGSTFS 17A3 271 EVQLVESGGGLV 546 FDNN 821 WYRQAPGK 1096 TIAHDGSTNYA 1371 QAGGSLSLSCAA QRELVA NSVKG SATLHR 27B7 272 EVQLVESGGGLV 547 SYAMS 822 WVRQAPGK 1097 AISSGGGSITTY 1372 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFS 17A6 273 EVQLVESGGGLV 548 SYAMS 823 WVRQAPGK 1098 AISSGGGSITTY 1373 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFS 17D7 274 EVQLVESGGGLV 549 YCAIG 824 WFRQAPGK 1099 CISSSDGSTYY 1374 QPGGSLRLSCAA EREGVS ADSVKG SGFTLD 46D4 275 EVQLVESGGGLV 550 DYAMS 825 WVRQAPGK 1100 SINWSGTHTDY 1375 QPGGSLRLSCAA GLEWVS AEDMKG SGFIFD 27B3 276 EVQLVESGGGLV 551 IRTMA 826 WYRQPPGN 1101 TIGSNGFATYP 1376 QAGGSLTLSCTAS QREWVA DSVKG ETTVR 27E5 277 EVQLVESGGGLV 552 IRTMA 827 WYRQPPGN 1102 TIGSNGFATYP 1377 QAGGSLTLSCTAS QREWVA DSVKG ETTVR 27D6 278 EVQLVESGGGLV 553 IRTMA 828 WYRQPPGN 1103 TIGSNGFATYP 1378 QAGGSLTLSCTAS QREWVA DSVKG ETTVR 30D10 279 EVQLVESGGGLV 554 IRTMA 829 WYRQPPGN 1104 TIGSNGFATYP 1379 QAGGSLTLSCTAS QREWVA DSVKG ETTVR 47G11 280 EVQLVESGGGLV 555 PMG 830 WFRQAPGK 1105 AIGSGDIITYYA 1380 QPGGSLRLSCAA EREFVA DSVKG SGRIFY 27C3 281 EVQLVESGGGLV 556 DYATS 831 WVRQAPGK 1106 AINSGGGSTYY 1381 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 11A101/1- 282 EVQLVESGGGLV 557 AMG 832 WFRQAPGK 1107 AISRSPGVTYY 1382 120 QAGGSLRLSCAA EREFVA ADSVKG SGRTFN 11A22/1- 283 EVQLVESGGGLV 558 SYAMA 833 WFRQAPGT 1108 GIRWSDGSTY 1383 122 QAGGSLRLSCAA EREFIA YADSVKG SGRTFS 12D44/1- 284 KVQLVESGGGLV 559 SYAMA 834 WFRQAPGT 1109 GIRWSDGSTY 1384 122 QAGGSLRLSCAA EREFIA YADSVKG SGRTFS 12E11/1- 285 EVQLVESGGGLV 560 SYAMA 835 WFRQAPGK 1110 GIRWSDGSTY 1385 122 QAGGSLRLSCAA EREFVG YADSVKG SGRTFS 13G111/ 286 EVQLVESGGGLV 561 SYAMG 836 WFRQAPGK 1111 AIRWSGGNTY 1386 1-123 QAGGSLRLSCAA ERAFVA YADSVKG SGRTFS 13F71/1- 287 EVQLVESGGGLV 562 NYALA 837 WFRQAPGK 1112 AINWRSGGST 1387 123 QAGGSLRLSCVA EREFVA YYADSVKG SGRTFS 14H61/1- 288 EVQLVESGGGLV 563 RFAMG 838 WFRQAPGK 1113 AVRWSDDYTY 1388 122 QAGGSLRLSCAA EREFVA YADSVKG SGRTFS 22B12/1- 289 EVQLVESGGGLV 564 SYAMA 839 WFRQAPGK 1114 GINKSGGITHS 1389 124 QAGGSLRLSCAA EREFVA ADSVKG SGRTFS 14H71/1- 290 EVQLVESGGGLV 565 SLTMA 840 WFRQAPGK 1115 NIKWSGDRIVY 1390 123 QAGGSLRLSCEA EREFVA ADSVKG SGLTIS 12D51/1- 291 EVQLVESGGGLV 566 IKSMG 841 WYFRQAPGK 1116 VIISSGTTTYAD 1391 120 QPGGSLRLSCAA QRELAA SVKG SGSAFS 11A111/1- 292 EVQLVESGGGLV 567 SSLMG 842 WFRQAPGK 1117 AITDNGGSTYY 1392 126 QAGGSLGLSCAA EREFVA ADSVKG AGRTFS 13G71/1- 293 EVQLVESGGGLV 568 SYAMG 843 WFRQAPGK 1118 AITSSGSTNYA 1393 124 QAGGSLRLSCAA ERDFVA DSVKG SGRAFS 13G74/1- 294 EVQLVESGGGLV 569 TYASMG 844 WFRQTPGK 1119 AITSSGSTNYA 1394 125 QAGGSLRLSCAT EREFVA DSVKG SGRTFS 11A71A/ 295 EVQLVESGGGLV 570 IITMG 845 WYRQRPGK 1120 TINSGGDTNYA 1395 1-116 QPGGSLRLSCAA PREWVG GSVKG SGNIDG 22B101/1- 296 EVQLVESGGGLV 571 DYAIG 846 WFRQAPGK 1121 AISSSGISTIYG 1396 123 QTGGSLRLSCAA EREFVA DSVKG SGPTFS 11B42/1- 297 EVQLVESGGGLV 572 NHIMG 847 WFRQAPGK 1122 HITWNGGSTYY 1397 123 QAGDSLRLSCAA ERELIA ADSVKG SGFTFS 13E111/1- 298 EVQLVESGGGLV 573 DYAIG 848 WFRQAPGK 1123 AISGWSGGTT 1398 124 QAGSSLRLSCALS EREFVA NYADSVKG GRTFS 14H12/1- 299 EVQLVESGGGLV 574 SAGVG 849 WFRQAPGK 1124 AISWNGVTIYY 1399 125 QAGGSLRLSCIAS ERDFVA ADSVKG ERTFS 13G101/ 300 EVQLVESGGGLV 575 RSRVA 850 WFRQAPGK 1125 VISGVGTSYAD 1400 1-123 QPGDSLRLSCSA EREFVT SVKG SEGTLS 13G41/1- 301 EVQLVESGGGLV 576 ADVMG 851 WYRQAPGK 1126 SISSGSAINYAD 1401 121 QPGGSLTLSCVG QREFVA SVKG SGRRFS 22B910/1- 302 EVQLVESGGGLV 577 MNDMG 852 WYRQAPGK 1127 TLTSAGNTNYA 1402 121 QPGGSLPLSCAA ORERVA DSVKG SGSIFR 21A81/1- 303 EVQLVESGGGLA 578 GMA 853 WFRRAPGK 1128 GIAWNGASIGS 1403 122 QAGGSLRLSCAV EREFVA ADSVRG FGRSRY 21A92/1- 304 EVQLVESGGGQV 579 TRAMG 854 WFRQAPEK 1129 GITMSGFNTRY 1404 127 QAGGSLRLSCTE EREFVA ADSVKG SGRAFN 22C712/1- 305 EVQLVESGGGLV 580 NYAMG 855 WFRQAPGK 1130 GISWSGGHTF 1405 123 QAGGSLGLSCAA EREFVA YADSVKG SGRTFS 11A13/1- 306 EVQLVESGGGLV 581 SYAMG 856 WFRQAPGK 1131 TIDWSGDTAFY 1406 125 QAGDSLRLSCVA EREFVA ADSVKG SGGTFG 13G93/1- 307 EVQLVESGGGLA 582 AYAMG 857 WFRQAPGK 1132 AVSWDGRNTY 1407 123 QAGDSLRLSCVD EREFVA YADSVKG SGSSFS 12C52/1- 308 EVQLVESGGGLV 583 SDTMA 858 WFRQAPGK 1133 RVSWIRTTYYS 1408 118 QAGGSLRLSCAV EREFVA DSVKG SGGTFE 12C61/1- 309 EVQLVESGGGLV 584 SNAMA 859 WFRQAPGN 1134 AIGWSGASTYY 1409 126 QAGGSLRLSCAA ERELVS IDSVEG SGRTFS 21A61/1- 310 EVQLVESGGGLV 585 TYTMG 860 WFRQAPGK 1135 AIRWSGGTTFY 1410 125 QAGDSLRLSCVA EREFVA GDSVKG SGDSFN 11A121/1- 311 EVQLVESGGGLV 586 SYSMG 861 WFRQAPGK 1136 AITWNGTRTYY 1411 126 QAGGSLRLSCVV DREFVS RDSVKG SEGTFS 11A91/1- 312 EAQLVESGGGLV 587 TTMG 862 WFRQAPGK 1137 AIRWSGGSAFY 1412 124 QAGGSLRLSCTA EREFVA ADSVKG SGRTYS 13G72/1- 313 EVQLVESGGGLV 588 SYAMG 863 WFRQAPGK 1138 AITSSGSTNYA 1413 118 QAGGSLRLSCAA ERDFVA DSVKG SGRAFS 13E81/1- 314 EVQLVESGGGLV 589 VYHMA 864 WFRQAPGK 1139 AIRSSGGLFYA 1414 124 QAGGSLRLSCAA EREFVA LSVSG SGGTFS 11B31/1- 315 EVQLVESGGGLV 590 VYHMG 865 WFRQAPGK 1140 AIRSGGTTLYE 1415 124 QAGGSLRLSCAA EREFVA DSVKG SGGAFG 13G81/1- 316 EVQLVESGGGLV 591 VYHMG 866 WFRQAPGK 1141 VIRSGGTTLYA 1416 124 QAGGSLRLSCAA EREFVA DSVKG SGGTFG 21A53/1- 317 EVQLVESGGGLV 592 VYHMG 867 WFRQAPGK 1142 AIRSGGTTLYE 1417 124 QAGGSLELSCAA EREFVA DSVKG SGGAFG 14H51/1- 318 EVQLVESGGGLV 593 VYTMA 868 WFRQAPGK 1143 AIRSGATTLYE 1418 124 QAGGSLRLSCAA EREFVA DSVKG SGGTFG 21A21/1- 319 EVQLVESGGGLV 594 VYHMG 869 WFRQAPGT 1144 VIRSGGTTLYE 1419 124 QAGGSLRLSCAA EREFVA DSVKG SGGTFG 21A111/1- 320 EVQLVESGGGLV 595 PYTMA 870 WFRQAPGK 1145 VTRSGGTTFYA 1420 124 QAGGSLKLSCAV EREFVA DSAKG SGRTIV 22B1212/ 321 EVQLVESGGGLV 596 SYAMS 871 WVRQAPGK 1146 AINSGGGSTSY 1421 1-122 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFS 11A31/1- 322 EVQLVESGGGLV 597 SGVMA 872 WFRQSPGE 1147 LITRNGETKKT 1422 120 QAGGSLRLSCAA EREFLA ADSVKG SGGTFS 13E51/1- 323 EVQLVESGGGLV 598 GYAMG 873 WFRQAPGK 1148 AIRWSGGITYY 1423 128 QAGGSLRLSCAA EREFVA ADSVKG SRHTFS 12D121/1- 324 EVQLVESGGGLV 599 TYGMG 874 WFRQAPGK 1149 AISRSGTGTYY 1424 126 QTGGSLRLSCAA AREFVA AGSMKG SGRAFS 13F121/1- 325 EVQLVESGGGLV 600 DYTMG 875 WFRQTPGK 1150 RVWWNGGSA 1425 119 QAGGSLRLSCAA EREFVA YYADSVKG SGRSFN 13G121/ 326 EVQLVESGGGLV 601 SAAMG 876 WFRQAPGK 1151 AISPIGSSKYYA 1426 1-127 RAGTSLRLSCADS EREFVS DSVKG ARTFS 22B41/1- 327 EVQLVESGGGLV 602 GDVIG 877 WFRQAPGK 1152 AISTSGGGTDS 1427 124 QPGGSLRLSCTV EREFVA ADSVKG FGRTFS 12D71/1- 328 EVQLVESGGGLV 603 TMG 878 WFRQAPGK 1153 AITWSGDSTNF 1428 125 QAGGSLGLSCAA EREFVT ADSVKG SGRTVS 13F42/1- 329 EVQLVESGGGLV 604 TTGVG 879 WFRQAPGK 1154 TIFVGGTTYYS 1429 111 QAGGSLRLSCVA GRESVA DSVKG SGRTLS 12C101/1- 330 EVQLVESGGGLV 605 TTGVG 880 WFRQAPGK 1155 TIFVGGTTYYS 1430 111 QAGGSLRLSCVA ERESVA DSVKG SGRTLS 14H91/1- 331 EVQLVESGGGLV 606 RDVMG 881 WFRQAPGK 1156 AKTWSGASTY 1431 127 QAGGSLRLSCAA EREFVA YADSVRG SGRTFS 13F41/1- 332 EVQLVESGGGLV 607 TTGVG 882 WFRQAPGK 1157 TIFVGGTTYYS 1432 111 QAGGSLRLSCVA ERESVA DSVKG SGRTLS 14H21/1- 333 EVQLVESGGGLV 608 SYHIG 883 WFRQAPGN 1158 AITWNGASTSY 1433 125 QAGGSLRLSCVR EREFVA ADSVKG SGGYFG 22B610/1- 334 EVQLVESGGGLV 609 INAMG 884 WYRPAPGK 1159 RITSTGSTNYA 1434 120 QPGGSLRLSCAA QRELVA DSVKG SGSIFS 12C32/1- 335 EMQLVESGGGLV 610 TYTMA 885 WFRQAPGK 1160 AIKSSDNSTSY 1435 127 QAGGSLRLSCAT EREFVV RDSVKG SERTFS 12D61/1- 336 EVQLVESGGGLV 611 PNVVG 886 WYRQAPGK 1161 AVTSGGITNYA 1436 116 QPGGSLRLSCAA QRELVA DSVKG SRSIFS 13G31/1- 337 EVQLVESGGGLV 612 RYKMG 887 WFRQAPGK 1162 ASRWSGGIKY 1437 125 QAGGSLRLSCAA EREFVA HADSVKG SGGTFS 22C65/1- 338 EVQLVESGGGLV 613 SYAMG 886 WFRQAPGK 1163 AIRWSGSATDY 1438 124 QAGGSLRLSCAV EREFVA SDSVKG SGFLFD 11A71/1- 339 EVQLVESGGGLV 614 VSVMG 889 WYRLAPGN 1164 TITADGITNYAD 1439 125 QPGGSLRLSCAA QRELVA SVKG SRSIRS 11B91/1- 340 EVQLVESGGALV 615 INTMG 890 WYRQAPGN 1165 AVTEGGTTSYA 1440 125 QPGGSLRLSCAA QREFVA ASVKG SGSIRS 11A81/1- 341 EVQLVESGGALV 616 INIMG 891 WYRQAPGK 1166 AVTEDGSINYA 1441 125 QPGGSLRLSCAA QREFVA ESVKG SDSIRS 11B121/1- 342 EVQLVESGGGLV 617 INTMG 892 WYRQAPGE 1167 EITEGGIINYTD 1442 127 QPGGSLRLSCAA QRELVA SVKG SGSSAS 12D31/1- 343 EVQLVESGGGLV 618 FNDMG 893 WYRQGPGK 1168 LINVGGVAKYE 1443 115 QPGGSLRLSCAA EREFVA DSVKG SRNIFD 11B51/1- 344 EVQLVESGGGLV 619 GRGMG 894 WFRQAPGK 1169 AVSWSGGNTY 1444 127 QAGGSLRLSCAA EREFVA YADSVKG SGGTFS 13G51/1- 345 EVQLVESGGGLV 620 GRAVG 895 WFRQAPGE 1170 GISWSGGSTD 1445 127 QAGGSLSLSCAA EREFVT YADSVKG SGGTFN 13F82A/1- 346 EVQLVESGGGLV 621 GRAMG 896 WFRQAPGK 1171 FVAATSWSGG 1446 130 QAGGSLRLSCAIS EREFRE SKYVADSVTG GRTLS 13E101/1- 347 EVQLVESGGGLV 622 NDHMG 897 WFRQAPGT 1172 ATGRRGGPTY 1447 128 QAGGSLRLSCAV ERELVA YADSVKG SGRTFN 22B85/1- 348 EVQLVESGGGLV 623 INAMG 898 WYRQAPGN 1173 TITGSTGTTYA 1448 120 RPGGSLRLSCAT QRELVA DSVKG SGSDIG 11B12/1- 349 EVQLVESGGGLV 624 NYAMG 899 WFRQAPGK 1174 AINWSGSHTDY 1449 118 QAGGSLRLSCAA EREFVS GDSVKG SGRALI 13G61/1- 350 EVQLVESGGGVV 625 SYVMG 900 WVRQAPGK 1175 GITRNSGRTRY 1450 118 QAGGSLRLSCAP AREFVA ADSVKG SGRTFS 14H41/1- 351 EVQLVESGGGLV 626 SYVMG 901 WVRQAPGK 1176 GITRNSIRTRYA 1451 118 QAGGSLRLSCAP AREFVA DSVKG SGRTFS 11B81/1- 352 EVQLVESGGGLV 627 NYIMG 902 WFRQALGQ 1177 AINRNGATAAY 1452 126 QAGGSLRLSCAA GREFVA ADSVKG SGRPVN 11C11/1- 353 EVQLVESGGGLV 628 AYAMG 903 WFRQAPGK 1178 TIRWTGGSSST 1453 121 QAGGSLRLSCAA ERESVA SYADSVKG SGRTFS 12D92/1- 354 EVQLVESGGGLV 629 MA 904 WFRQAPGK 1179 AMNWSGGSTK 1454 123 QAGGSLRLSCAA EREFVA YAESVKG SGRTYN 13E61/1- 355 EVQLVESGGGLV 630 MG 905 WFRQAPGK 1180 AISWSQYNTKY 1455 123 QAGGSLRLSCTA EREFVA ADSVKG SGQTFN 22B71/1- 356 EVQLVESGGAFV 631 FNVMG 906 WYRQGPGQ 1181 SITYGGNINYG 1456 114 QPGGSLRLSCAA QLELVA DPVKG SGSDVW 21A121/1- 357 EVQLVESGGGLV 632 MG 907 WFRQAPGK 1182 GVNWGGGSTK 1457 123 QAGGSLRLSCTA EREFVA VADSVKE SGRAFN 13F101/1- 358 EVQLVESGGGLV 633 DLHMG 908 WFRQAPGK 1183 FTRWPSITYIAE 1458 124 QAGGSLRLSCQL EREFVG HVKG SGGTVS 11A43/1- 359 EVQLVESGGGLV 634 VNHMG 909 WYRQAPGK 1184 AITSDHITWYA 1459 123 QAGGSLRLSCAA QREFVA DAVKG SGSIFR 12C81/1- 360 EVQLVESGGGLV 635 DNTMA 910 WYRQAPGN 1185 TINVGGGTYYA 1460 117 QPGGSLRLSCAG QRDLVA GPVKG SGNIVR 11B21/1- 361 EVQLVESGGGLV 636 MYLMG 911 WFRQAPGK 1186 TINRRGGNTYY 1461 124 QAGGSLRLSCAA EREFVS ADSVKG SGRTFS 11B71/1- 362 EVQLVESGGGLV 637 RYAMG 912 WFRQAPGK 1187 TISWSGGRDTV 1462 126 QAGGSLRLSCAA EREFVA YADSVKG SGRTFE 12C121/1- 363 EVQLVESGGDLV 638 YSGIG 913 WFRQAPEK 1188 CIESGDGTTTY 1463 126 QPGESLRLSCAV EREAVS VDSVKG SGVTVD 22C51/1- 364 EVQLVESGGGLV 639 RYDIG 914 WFRQAPGK 1189 AINWSGGTTSF 1464 121 QAGASLRLSCAA GREFVA GDSVKG SGRTFS 12D11/1- 365 EVQLVESGGGLV 640 GSRMG 915 WFRQAPGK 1190 AIRWSGGITWY 1465 123 QTGGSLRLSCAA EREFVA AESVKS SGRTFS 12D14/1- 366 EVQLVESGGGLV 641 GSRMG 916 WLRQAPGK 1191 AVRWSGGITW 1466 123 QAGGSLRLSCAA EREFVA YAESVKG SGRTFS 12C111/1- 367 EVQLVESGGGLV 642 SYAMG 917 WFRQAPGK 1192 TISRSGGRTSY 1467 123 QAGGSLRLSCAV VREFVA ADSVKG SGLTFS 22B55/1- 368 EVQLVESGGGLV 643 TYAMG 918 WFRQAPGK 1193 TISRSGGRTSY 1468 123 QAGGSLRLSCAV VREFVA ADSVKG SGLTFS 14H121/1- 369 EVQLVESGGGLV 644 FKAMG 919 WFRQGPGK 1194 RIAGGSTNYAD 1469 113 QPGGSLRLSCAA RRELVA SVKG SGITFR 12C71/1- 370 EVQLVESGGGLV 645 SYALG 920 WFRQSPGK 1195 AIDWDGSRTQ 1470 125 QAGGSLRLSCTA ERESVA YADSVKG SGGTFG 21A31/1- 371 EVQLVESGGGLV 646 SVAMG 921 WFRQGPGK 1196 TITWSGDSTYV 1471 125 QAGGSLRLSCAA EREFAA TDSVKG SEPTFS 12C91/1- 372 EVQLVESGGGLV 647 YYHMA 922 WFRQAPGK 1197 AINLSSGSTYY 1472 121 QAGGSLRLSCVA EREFIA PDSVKG SGRTSS 14H81/1- 373 EVQLVESGGGLV 648 NYRMA 923 WFRQAPRK 1198 AISRSGESTYF 1473 125 QAGGSLSLSCAA EREFVA ADSMKG SGRTFS 12C42/1- 374 EVQLVESGGGLV 649 RYAMH 924 WFRQAPGS 1199 GISWDGGSTF 1474 124 QAGGSLRLSCAA ERDFVA YANSVKG SGRTFS 12D102/1- 375 EVQLVESGGGLV 650 FNAMG 925 WSREAPGK 1200 RIISDDSTLYAD 1475 118 QPGGSLRLSCAA RRELVA SVKG SGSSLS 11A52/1- 376 EVQLVESGGGLV 651 NYAMR 926 WFRQAPGK 1201 TINWSGSHTDY 1476 120 QAGGSLRLSCAA EREFVA ADSVKG SGRALS 14H111/1- 377 EVQLVESGGGLV 652 SFAMR 927 WFRQAPGK 1202 AINWSGTHTDY 1477 120 QAGGSLRLSCAA EREFVA ADSVKG SGRALI 11B61/1- 378 EVQLVESGGGLV 653 GYGMG 928 WFRQAPGK 1203 AVGWYGSTYF 1478 120 QAGGSLRLSCAA EREFVA ADSVKG SGRTSS 12E42/1- 379 EVQLVESGGGLV 654 LRTMG 929 WYRQAPGN 1204 LISAGDSTYYP 1479 118 QPGGSLRLSCAH QRELVA DSVKG SGRAFS 13F81A/1- 380 EVQLVESGGGLV 655 RYAMG 930 WFRQAPGK 1205 AISWTGGSSYY 1480 128 QAGGSLRLSCAA EREFVA GDSVKG SGRTFS 11B102/1- 381 EVQLVESGGGLV 656 SHAIS 931 WFRQAPGK 1206 AINWSGSHRD 1481 118 QAGGSLRLSCAA AREFVA YADSAKG SGGIFS 21A41/1- 382 EVQLVESGGGLV 657 NYAWS 932 WFRQAPGK 1207 AINWSGGYTD 1482 120 QAGGSLRLSCAA ERGFVA YADSVKG SGRIFS 14H101/1- 383 EVQLVESGGGLV 658 SSPMG 933 WFRQAPGK 1208 ATTRSGGLPYY 1483 128 QAGGSLRLSCAA EREVVA SDSVKG SGRTFI 12E21/1- 384 EVQLVESGGGLV 659 IHVVG 934 WYRKAPGK 1209 YIGTAGATHYA 1484 115 QPGGSLRLSCAA QREVVA DSVKG SGSIDS 13F21/1- 385 EVQLVESGGGLV 660 INATS 935 WYRQAPGN 1210 TIIGDGRTHYA 1485 123 QSGGSLRLSCVA QRELVA DSVKD SGTIVS 12E33/1- 386 EVQLVESGGGMV 661 NYGMG 936 WFRQAPGK 1211 SINWSGTHTYD 1486 119 QAGGSLRLSCAA EREFVS ADFVKG SGLTLS 13G11/1- 387 EVQLVESGGGLV 662 SNYAMG 937 WFRQAPGK 1212 TINWSGSHSDY 1487 122 QAGGSLRLSCAA EREFVA ADSVKG SGRTFI 118N121_A6_2_OK/ 388 EVQLVESGGGLV 663 GYSVG 938 WFRQSPGK 1213 GINWSGRTYY 1488 1-123 QAGGSLRLSCVA EREFVG VDSVKG SGRTFS 118N121_B8_1_OK/ 389 EVQLVESGGGLV 664 FASYAMG 939 WFRQAPGK 1214 AIRGSGGSTYI 1489 1-135 QDGGSLRLSCAA AREFVA ADPARSTYYAD SGQLAN FVKG 118N121_A2_2_OK/ 390 KVQLVESGGGLV 665 NYSVG 940 WFRQAPGK 1215 ALSKDGARTYY 1490 1-124 QAGGSLRLSCAA EREFVA AASVKG SGRTFS 118N121_A8_2_OK/ 391 EVQLVESGGGLV 666 SHAMG 941 WFRQAPGE 1216 TIRWSGSATFY 1491 1-124 QAGGSLTLSCVIS EREFVA SDSVKG GLTLE 118N121_B3_1_OK/ 392 EVQLVESGGGLV 667 DLALG 942 WFRRAPGK 1217 AISSSGVTTIYA 1492 1-123 QPGGSLRLSCAA EREHVA DSVRG SGRTFS 118N121_A5_2_OK/ 393 EVQLVESGGGSV 668 TNAMG 943 WHRQVSGK 1218 IVTDGFTNYAD 1493 1-114 QPGGSLRLSCVA ERELVA FAKG SGSISS 118N121_A9_2_OK/ 394 EVQLVESGGGSV 669 VNAMG 944 WHRQVPGK 1219 IVTDGFTNYAD 1494 1-114 QPGGSLRLSCVA QRELVA FAKG SGSISS 118N121_A7_1_OK/ 395 EVQLVESGGGLV 670 IDVMG 945 WHRQAPGK 1220 DISFGGNTNYA 1495 1-122 QPGGSLRLSCAA ERELVS NSVKG SGNIKS 118N121_A10_1_OK/ 396 EVQLVESGGGLV 671 DYAIG 946 WFRQAPGK 1221 CIANSEGTKYY 1496 1-131 QAGGSLRLSCAA EREGVS ADSAQG SGFSFA 118N121_A11_1_OK/ 397 EVQLVESGGGLV 672 MYGMR 947 WVRQAPGK 1222 SINSDGDTTYY 1497 1-120 QPGGSLRLSCAA GPERVS ADSVKG SGFPFG 118N121_B7_4_OK/ 398 EVQLVESGGGLE 673 SAAMG 948 WFRQAPGK 1223 AISRDGAATYY 1498 1-124 QAGGSLRLSCAA EREFVA TDSVKG SGLTFR 118N121_B2_1_OK/ 399 EVQLVESGGGLV 674 DRAIA 949 WFRQAPGK 1224 CITPHHGGIIFT 1499 1-130 QAGGSLRLSCAA AREGVS RESVKG SGFSLD 118N121_B7_1_OK/ 400 EMQLVESGGGLV 675 INAMA 950 WYRQAPGN 1225 AVTSGGGTNY 1500 1-119 QPGGSLRLSCAA ERELVA ATSVKG SGNIPP Clone FR3 ID CDR 3 ID FR4 13D11 RFTISRDNAKNTLFLQMNS 1501 GWKIVPTNP 1776 RGHGTQVTVSS LNPEDTAVYYCGQ 2B4 RFTISRDNAKNTLFLQMNS 1502 GWKIRPTIP 1777 MGHGTQVTVSS LSPEDTAVYYCNQ 2G2 RFTISRDNAKNTLFLQMNN 1503 GWKIVPTDL 1778 GGHGTQVTVSS LTPEDTAVYYCNR 13D2 RFTISRDNAKNTLFLQMNN 1504 GWKIVPTDR 1779 GGHGTQVTVSS LRSEDTAVYSCNQ 2D5 RFTISRDNAKNTLFLQMNS 1505 GWKIVPTDR 1780 GGHGTQVTVSS LRSEDTAVYYCNQ 2F4 RFTISRDNAKNTLFLQMNS 1506 GWKIVPTDR 1781 RGHGTQVTVSS LRSEDTAVYYCNQ 2C3 RFTISRDNAKNTLFLQMNS 1507 GWKIVPTDR 1782 TGHGTQVTVSS LRSEDTAVYYCNQ 17E3 RFTISRDNAKNTVYLEMNS 1508 GWKIVPTDR 1783 TGHGTQVTVSS LTPEDTAVYYCNQ 17H3 RFTISRDNAKNTLFLQMNS 1509 GWKIVPTDR 1784 GGHGTQVTVSS LRSEDTAVYYCNQ 17D2 RFTISRDNAKNTLFLQMNS 1510 GWKIVPTDR 1785 GSHGTQVTVSS LRSEDTAVYYCNQ 2F1 RFTISRDNAKNTLFLQMNS 1511 GWKIVPMDR 1786 RGHGTQVTVSS LTPEDTAVYYCNQ 2E2 RFTISRDNAKNTLFLQMNS 1512 GWKIIPTDR 1787 RGHGTQVTVSS LTPEDTAVYYCNQ 2C2 RFTISRDNARNTLFLQMNS 1513 GWKILPTDR 1788 RGHGTQVTVSS LTPEDTAIYYCNQ 2E3 RFTISRDNAKNTLFLQMNS 1514 GWKILPTNR 1789 GSHGTQVTVSS LSPEDTAVYYCNQ 13B10 RFTISRDNAKNTLFLQMNS 1515 GWKILPTNR 1790 GSHGTQVTVSS LSPEDTAVYYCNQ 2D1 RFTISRDNAKNTLFLQMNS 1516 GWKILPTNR 1791 GSHGTQVTVSS LSPEDTAVYYCNR 2H3 RFTISRDNAKNTLFLQMNS 1517 GWKIIPTDR 1792 RGHGTQVTVSS LTPEDTAVYYCNQ 2H1 RFVISRDNAKNTLFLQMNS 1518 GWKIIPTDR 1793 RGHGTQVTVSS LSPEDTAVYYCNQ 2C1 RFTISRDNAKNTLFLQMNS 1519 GWKIIPTDR 1794 RGHGTQVTVSS LTPEDTAVYYCNQ 15C5 RFTISRDNAKNTLFLQMNS 1520 GWKIIPTDR 1795 RGHGTQVTVSS LTPEDTAVYYCNQ 2B3 RFTISRDNAKNTLFLQMNS 1521 GWKIIPTDR 1796 RGHGTQVTVSS LTPEDTAVYYCNQ 29H2 RFTISRDNAKNTLFLQMNN 1522 GWKIIPTDR 1797 RGHGTQVTVSS LTPEDTAVYYCNQ 17E4 RFVISRDNAKNTLFLQMNS 1523 GWKIIPTDR 1798 RGHGTQVTVSS LSPEDTAVYYCNQ 17A2 RFTISRDNAKNTLFLQMNS 1524 GWKVWPTDR 1799 GTHGTQVTVSS LSPEDTAVYYCNK 15D1 RFTISRDNAKNTLFLQMNS 1525 GWKVWPTDR 1800 GTHGTQVTVSS LNPEDTAVYYCNQ 17B8 RFTISRDNAKNTLFLQMNS 1526 GWKILPAER 1801 RGHGTQVTVSS LTPEDTAVYYCNQ 15C11 RFTISRDNAKNTLFLQMNS 1527 GWKILPAER 1802 RGHGTPVTVSS LTPEDTAVYYCNQ 15G8 RFTISRDNAKNTLFLQMNS 1528 GWKILPAER 1803 RGHGTQVTVSS LTPENTAVYYCNQ 17H4 RFTISRDNAKNTLFLHMNN 1529 GWKIHPADR 1804 GGHGTQVTVSS LSPEDTAVYYCGQ 27G8 RFTISRDNAKNTLFLQMNS 1530 GWKILPAER 1805 RGHGTQVTVSS LTPEDTAVYYCNQ 38C6 RFTISRDNAKNTLFLQMNS 1531 GWKIRPTIP 1806 MGHGTQVTVSS LSPEDTAVYYCNQ 2A4 RFTISRDNAKKTVYLQMNS 1532 GWQSTTKN 1807 WGQGTQVTVSS LQSEDTAVYYCGT QGY 15G7 RFTISRDNNKKTVYLQMN 1533 GWQSTTKN 1808 WGQGTQVTVSS SLKSEDTAVYYCAT QGY 15B7 RFTISRDNAKKTVYLQMNS 1534 GWQSTTKS 1809 WGQGTQVTVSS LQSEDTAVYYCGT QGY 5G4 RFTISRDNAKKTLYLQMNS 1535 GWQSTTKN 1810 WGQGTQVTVSS LKSEDTAVYYCAT QNY 13B2 RFTISRNNANNTLYLQMN 1536 NWRDAGTT 1811 AGQGTQVTVSS NLKFEDTAVYYCAK WFEKSGS 2E5 RFTISRNNANNTLYLQMN 1537 NWRDAGTT 1812 AGQGTQVTVSS NLKFEDTAVYYCAK WFEKSGS 15G1 RFTISRNNANNTLYLQMNS 1538 NWRDAGTT 1813 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 27B1 RFTISRNNANNTLYLQMN 1539 NWRDAGTT 1814 AGQGTQVTVSS NLKFEDTAVYYCAK WFEKSGS 17E7 RFTISRNNANNTLYLQMNS 1540 NWRDAGTT 1815 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 17D8 RFTISRNNANNMLYLQMN 1541 NWRDAGTT 1816 AGQGTQVTVSS SLKSEDTAVYYCAK WFEKSGS 5F8 RFTISRNNANNTLYLQMN 1542 NWRDAGTT 1817 AGQGTQVTVSS NLKFEDTAVYYCAK WFEKSGS 2D4 RFTISRNNANNTLYLQMNS 1543 NWGDAGTT 1818 AGPGTQVTVSS LKSDDTAVYYCAK WFEKSGS 13D8 RFTISRNNANNTLYLQMNS 1544 NWGDAGTT 1819 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 17G8 RFTISRNNANNTLYLQMNS 1545 NWGDAGTT 1820 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 2H4 RFTISRNNANNTLYLQMNS 1546 NWGDAGTT 1821 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 2F3 RFTISRNNANNTLYLQMNS 1547 NWGDAGTT 1822 AGPGTQVTVSS LKSDDTAVYYCAK WFEKSGS 2F5 RFTISRNNANNTLYLQMNS 1548 NWGDAGTT 1823 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 30E10 RFTISRNNANNTLYLQMNS 1549 NWGDAGTT 1824 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 29H1 RFTISRNNANNTLYLQMNS 1550 NWGDAGTT 1825 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 17E2 RFTISRNNANNTLYLQMNS 1551 NWGDAGTT 1826 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 2B1 RFTISRNNANNTLYLQMNS 1552 NWGDAGTT 1827 AGPGTQVTVSS LKSDDTAVYYCAK WFEKSGS 2A5 RFTISRNNANNTLYLQMNS 1553 NWGDAGTT 1828 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 13C12 RFTISRNNANNTLYLQMNS 1554 NWGDAGTT 1829 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 17E10 RFTISRNNANNTLYLQMNS 1555 NWGDAGTT 1830 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 27D4 RFTISRNNANNTLYLQMNS 1556 NWGDAGTT 1831 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 15F9 RFTISRNNANNTLYLQMNS 1557 NWGDAGTT 1832 AGQGTQVTVSS LKSDDTAVYYCAK WFEKSGS 30H9 RFTISRNNANNTLYLQMNS 1558 NWGDAGTT 1833 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 39C1 RFTISRNNANNTLYLQMNS 1559 NWGDAGTT 1834 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 27G2 RFTISRNNANNTLYLQMNS 1560 NWGDAGTT 1835 AGPGTQVTVSS LKSDDTAVYYCAK WFEKSGS 2D3 RFTISRNNANNTLYLQMNS 1561 NWRDAGTT 1836 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 5F7 RFTISRDNAKNTVYLQMN 1562 FRTAAQGTDY 1837 WGQGTQVTVSS SLKPEDTAVYYCKR 118N121_A1_4_OK/ RFTISRDNAKNTLHLQMNS 1563 HKTPYTTLE 1838 WGQGTQVTVSS 1-127 LKPEDTAVYYCAY LNRPHAFGS 47D5 RFTISRVNAKDTVYLQMNS 1564 DQRLDGSTL 1839 WGQGTQVTVSS LKPEDTAVYYCYM AY 14B11 RFTISRDNAKKTVYLQMNS 1565 ETYGSGSSL 1840 WGQGTQVTVSS LKPEDTARYYCGV MTEYDY 14B10 RFTISRDNAKETVYLQMNS 1566 ETYGSGSSL 1841 WGQGTQVTVSS LKPDDTGVYYCAA MSEYDY 14B4 RFTISRDNAKETVYLQMNS 1567 ETYGSGSSL 1842 WGQGTQVTVSS LKPEDTGVYYCAA MSEYDY 14C11 RFTISRDNAKETVYLQMNS 1568 ETYGSGSSL 1843 WGQGTQVTVSS LKPDDTGVYYCAA MSEYDY 14B5 RFTISRDNAKETVYLQMNS 1569 ETFGSGSSL 1844 WGQGTQVTVSS LKPDDTGVYYCAA MSEYDY 14C6 RFTISRDNAKKTVYLQMNS 1570 DTYGSGSSL 1845 WGQGTQVTVSS LKPEDTAVYYCAT MNEYDY 14A4 RFTISRDNAKKTAYLQMNS 1571 ETYGSGSSL 1846 WGQGTQVTVSS LKPEDTAVYYCAA MNEYDY 14B3 RFTISRDNAKKTAYLQMNS 1572 ETYGSGSSL 1847 WGQGTQVTVSS LKPEDTAVYYCAA MNEYDY 14C1 RFTISRDNAKKTVYLQMNS 1573 ETYGSGSSL 1848 WGQGTQVTVSS LKPEDTAVYYCAT MNEYDY 14A12 RFTISRDNAKKTVYLQMNS 1574 EPYGSGSSL 1849 WGHGTQVTVSS LKPEDTAVYYCAA ISEYDY 14A2 RFTISRDNAKKTVYLQMNS 1575 EPYGSGSSL 1850 WGHGTQVTVSS LKPEDTAVYYCAA ISEYDY 14A1 RFTISRDNAKKTVYLQMNS 1576 EPYGSGSSL 1851 WGHGTQVTVSS LKPEDTAVYYCAA MSEYDY 17C3 RFTMARDNANHTVHLQM 1577 NWKMLLGV 1852 WGQGTQVTVSS NSLKPEDTAVYYCNA ENDY 46D3 RFTVSRDIAENTVYLQMNT 1578 KIGWLSIRG 1853 WGQGTQVTVSS LKSEDTAVYYCAA DEYEY 27H5 RFTISSDNAKNTVYLQMNS 1579 LPFVCPSGS 1854 WGQGTQVTVSS LKPEDTAVYYCAA YSDYGDEY DY 17C2 RFTISRDNAKNTLYLQMSS 1580 HVSDSDYTE 1855 WGQGTQVTVSS LKPEDTALYYCTK YDY 17D11 RFTISRDNAKNTLYLQMNS 1581 RRSSLYTSS 1856 WGQGTQVTVSS LKPEDTAVYYCAA NVFEYDY 15A6 RFTVSRDNANNTVYLQMN 1582 VNADY 1857 WGQGTQVTVSS TLKPEDTAVYYCRD 17B6 RFTVSRDNAKNTVYLQMN 1583 VNADY 1858 WGQGTQVTVSS SLKAEDTAVYYCWD 17C5 RFTVSRDNANNTVYLQMN 1584 VNADY 1859 WGQGTPVTVSS TLKPEDTAVYYCRD 15E11 RFTVSRDNDKNTLYLQMN 1585 VNADY 1860 WGQGTQVTVSS SLKPEDTAVYYCRD 15C2 RFTVSRDNANNTVYLQMN 1586 VNADY 1861 WGQGTQVTVSS TLKPEDTAVYYCRD 2A3 RFTVSRDNAKNTLYLQMN 1587 VNGDI 1862 WGQGTQVTVSS SLKPEDTATYLCRD 27A5 RFTVSRDNAKNTLYLQMN 1588 VNGDI 1863 WGQGTPVTVSS SLEPEDTATYYCWD 2C5 RFTVSRDNAKNTLYLRMN 1589 VNADI 1864 WGQGTQVTVSS SLKSEDSATYTCRA 27G5 RFTVSRDNAKNTVYLQMN 1590 VNGDY 1865 WGQGTQVTVSS SLKPEDTAVYYCRD 13A9 RFTVSRDNANHTVYLQMN 1591 VNGDY 1866 WGQGTQVTVSS SLKPEDTAVYYCRD 29E9 RFTVSRDNAKNTVYLQMN 1592 VNGDY 1867 WGQGTQVTVSS SLKPEDTAVYYCRD 15D8 RFTVSRDNAKNTVYLQMN 1593 VNGDY 1868 WGQGTQVTVSS SLKPEDTAVYYCRD 15G4 RFTVSRDNAKNTLYLQMN 1594 VNGDY 1869 WGQGTQVTVSS SLKPEDTAVYYCWD 15D12 RFAISRDNAKNTAYLQMN 1595 VWKLPRFV 1870 WGQGTQVTVSS SLKAEDTAVYYCNA DNDY 15E12 RFTMARDNANHTVHLQM 1596 NWKMLLGV 1871 WGQGTQVTVSS NSLKPEDTAVYYCNA ENDY 13D7 RFTMARDNANHTVHLQM 1597 NWKMLLGV 1872 WGQGTQVTVSS NSLKPEDTAVYYCNA ENDY 13A8 RFTISRDNAENTVYLQMN 1598 VNRDY 1873 WGQGTQVTVSS SLKPEDTAVYYCWD 15A4 RFTISRDNAKNTVYLQINS 1599 VNRDY 1874 WGQGTQVTVSS LKPEDTAVYYCWD 17F7 RFTISRDNAKKTMYLQMN 1600 VNRDY 1875 WGQGTQVTVSS SLKPDDTAVYYCRD 15C8 RFTVSRDNAENTVYLQMN 1601 VNRDY 1876 WGQGTQVTVSS SLKPEDTAVYYCRD 17A10 RFTLSRDNAKNTVYLQMN 1602 VNRDY 1877 WGQGTQVTVSS SLKPEDTAVYYCRD 27D3 RFTISRDNAENTVYLQMN 1603 VNRDY 1878 WGQGTQVTVSS SLKPEDTAVYYCWD 13B12 RFTLSRHNAENTVYLQMN 1604 VNRDY 1879 WGQGTQVTVSS SLKPEDTAVYYCRD 15B2 RFTISSDNAKSTIYLQMNS 1605 VNRDY 1880 WGQGTQVTVSS LKPDDTAVYYCRD 15B11 RFTISSDNAKNTIYLQMNS 1606 VNRDY 1881 WGQGTQVTVSS LKPDDTAVYYCRD 13C9 RFTISRDNAKKTVYLQMNS 1607 VNRDY 1882 WGQGTQVTVSS LKPEDTAVYYCRD 17D5 RFTISRDGATNTVFLQMN 1608 VNRDY 1883 WGQGTQVTVSS NLKPEDTAVYYCRD 27B5 RFTVSRDNAENTVYLQMN 1609 VNRDY 1884 WGQGTQVTVSS SLKPEDTAVYYCRD 27C7 RFTVSRDNADNTVYLQMN 1610 VNRDY 1885 WGQGTQVTVSS SLKPEDTAVYYCRD 13D4 RFTISSDNAKSTIYLQMNS 1611 VNREY 1886 WGQGTQVTVSS LEPDDTAVYYCRD 15G5 RFTISSDNAKKTIYLQMNS 1612 VNREY 1887 WGQGTQVTVSS LKPDDTAVYYCRD 13C4 RFTISSDNAKSTIYLQMNS 1613 VNREY 1888 WGQGTQVTVSS LKPDDTAVYYCRD 46G1 RFTISRDNAKNAVILQMNN 1614 KPGWVARD 1889 WGQGTQVTVSS LKTEDTAVYYCAA PSQYNY 46E4 RFTISRDNAKNAVILQMNN 1615 KPGWVARD 1890 WGQGTQVTVSS LKTEDTAVYYCAA PSEYNY 17B5 RFTISNDNAKDTVYLQMN 1616 FNRDL 1891 WGQGTQVTVSS SLKSEDTAVYYCRN 15C9 RFTISRDNAKNTVYLQMN 1617 VNRDL 1892 WGQGTQVTVSS SLKPEDTGVYYCRD 13D10 RFTISKDYTRNTVYLQVNS 1618 DANPNCGL 1893 WGQGTQVTVSS LKPEDTAVYHCAT NVWNS 17C6 RFTISGDTAKSTLYLQMNS 1619 RDYYYMPF 1894 WGQGTQVTVSS LKPEDTAMYYCYG 15A2 RFAISRDNAKNTVYLQMN 1620 INEDQ 1895 WGQGTQVTVSS SLKPEDTAVYYCRD 17A8 RFAISRDNAKNMVYLQMN 1621 INEDQ 1896 WGQGTQVTVSS TLKPEDTAVYYCRD 15G10 RFAISRDNAKNTVYLQMN 1622 INEDQ 1897 WGQGTQVTVSS SLKPEDTAVYYCRD 27A3 RFAISRDNAKNTVYLQMN 1623 INEDQ 1898 WGQGTQVTVSS TLKPEDTAVYYCRD 17H10 RFAISRDNAKNTVYLQMR 1624 INADQ 1899 WGQGTQVTVSS SLKPEDTAVYYCRD 30D10 RFTISRDNAKNTVYLQMN 1625 INRDI 1900 WGQGSQVTVSS SLKPEDTAVYYCRD 15H4 RFTISRDNAKNTVYLQMN 1626 INHDI 1901 WGRGSQVTVSS NLKPEDTAVYYCRD 17B7 RFTISRDNAKKTVYLQMNS 1627 INRDY 1902 WGQGTQVTVSS LKPEDTAVYFCRD 15D2 RFTFSKDGAKNTVYLQMN 1628 INRDY 1903 WGQGTQVTVSS SLKPEDTAVYYCRD 17G5 RFTISRDNAKNTVYLQMN 1629 INRDY 1904 WGQGTQVTVSS SLKPEDTAVYYCRD 15B6 RFTLSRDNAKKTVYLQMN 1630 INGDY 1905 WGQGTQVTVSS SLKPEDTAIYYCRD 27F2 RFTISISRDNVEYTVDLQM 1631 INGDY 1906 WGQGTQVTVSS DSLKPEDTAVYYCRD 17F5 RFTISRDNAKNTVYLQVNS 1632 TNGDY 1907 WGQGTQVTVSS LKPEDTAVYYCWD 17B2 RFTISRDNAKNTLYLQMNS 1633 DISTFGWGP 1908 WGQGTQVTVSS LKPEDTALYYCTK FDY 27H4 RFTISRDNAKNTVYLEMNS 1634 RWDIVGAIW 1909 WGQGTQVTVSS LKPEDTGVYYCNG 13A4 RFTISRDNAKNTVYLEMNS 1635 RWDIVGAIW 1910 WGQGTQVTVSS LKPEDTGVYYCNG 2A1 RFTISRDNTKNTLDLQMNS 1636 RWLEIGAEY 1911 WGQGTQVTVSS LKPEDTAVYYCHG 15E10 RFTISRDNAKNTVVLQMN 1637 SWSYPGLTY 1912 WGKGTLVTVSS GLKPEDTAVYYCKG 27E7 RFTISRDNAKNTVYLQMN 1638 VWRAIGRTY 1913 WGQGTQVTVSS DLKPEDTAVYYCNG 47E5 RFTIARDNAKNTVSLQMN 1639 DRRSYNGR 1914 WGQGTQVTVSS SLKPEDTAVYYCNL QY 2G4 RFTISRDNTKNTMYLQMV 1640 GGREYSGV 1915 WGQGTQVTVSS SLKPEDTAVYYCNV YYYREY 14D4 RFTISRDNAKNTVYLQMAS 1641 RPSDTTWS 1916 WGQGTQVTVSS LKPEDTAAYYCAA ESSAS 17A5 RFTISRDNVKNTLYLQMNS 1642 DLGNSGRG 1917 WGQGTQVTVSS LKSEDTAVYYCAK PYTN 15D10 RFTISRDNAKNTVYLQMN 1643 DCGVPGFG 1918 WGQGTQVTVSS SLKPEDTAVYYCAT WTFSS 13C2 RFTISRDNAKNAVHLQMN 1644 KQLPYLQNF 1919 WGQGTQVTVSS SLRLEDTAVYYCNA 17G11 RFTISRDNPETAVHLQMN 1645 KQLPYLQNF 1920 WGQGTQVTVSS SLKLEDTAVYYCNA 17A3 RFTISRDNARDTLFLQMHA 1646 HRWGLNY 1921 WGQGTQVTVSS LQPEDTAVYMCNL 27B7 RFTISRDNAKNTLYLQMSS 1647 ARSSSSYYD 1922 WGQGTQVTVSS LKPEDTALYYCAK FGS 17A6 RFTISTDNAKNTLYLQMSS 1648 ARSSSSYYD 1923 WGQGTQVTVSS LKPEDTALYYCAK FGS 17D7 RFTISRDNAKNTVYLQMN 1649 DRGSGTCY 1924 WGQGTQVTVSS SLKPEDTAVYYCAT ADFGS 46D4 RFTISRDNAKKTLYLQMNS 1650 GWGPAVTSI 1925 ATLGTQVTVSS LQSEDTAVYYCAK PV 27B3 RFTISRDNAKNTVYLQMN 1651 INRDI 1926 WGQGSQVTVSS SLKPEDTAVYYCRD 27E5 RFTISRDNAKNTVYLQMN 1652 INRDI 1927 WGQGSQVTVSS SLKPEDTAVYYCRD 27D6 RFTISRDNAKNTVYLQMN 1653 INRDI 1928 WGQGSQVTVSS SLKPEDTAVYYCRD 30D10 RFTISRDNAKNTVYLQMN 1654 INRDI 1929 WGQGSQVTVSS SLKPEDTAVYYCRD 47G11 RFTISRDNAKNTVYLQMN 1655 SRDYSRSR 1930 WGQGTQVTVSS SLKPEDTAVYYCAS DPTSYDR 27C3 RFTISRDNAKNTLYLQMNS 1656 PRGSSLYLL 1931 WGQGTQVTVSS LKPEDTAVYYCAR EYDY 11A101/1- RFTTSRDNAKNTVYLQMN 1657 DFYLATLAH 1932 WGQGTQVTVSS 120 DLKPEDTAVYYCAA EYDY 11A22/1- RFTISRDNAKNTVYLQMN 1658 DFYVSTLAH 1933 WGQGTQVTVSS 122 SLKPEDTAVYYCAA EYDY 12D44/1- RFTISRANAKNTVYLQMN 1659 DFYVSTLAH 1934 WGQGTQVTVSS 122 GLKPEDTAVYYCAA EYDY 12E11/1- RFTISRDNAKITVYLQMNS 1660 DFYVSTLAH 1935 WGQGTQVTVSS 122 LKPEDTAVYYCAA EYDY 13G111/ RFTISRDNAKNTVYLQMN 1661 DTFTLSTLS 1936 WGQGTQVTVSS 1-123 SLKPEDTAVYYCAA HEYDY 13F71/1- RFTISRDNAKNTVYLQMN 1662 DLIVATLPGE 1937 WGQGTQVTVSS 123 SLKPEDTAVYYCAA YDY 14H61/1- RFTISRDNAKNTVYLQMN 1663 DEILATLPHE 1938 WGQGTQVTVSS 122 SLSPEDTAVYYCAA YDY 22B12/1- RFTISRDNAKNTVYLQMN 1664 DAYTVIATLP 1939 WGQGTQVTVSS 124 SLKPEDTAVYYCAA HEYDY 14H71/1- RFTISRDSAKNAVNLQMEL 1665 KHSTVAGLT 1940 WGQGTQVIVSS 123 VESDDTAVYYCAA HEYDY 12D51/1- RFTISRDSAKNTVYLQMDS 1666 VYVSTWGN 1941 WGQGTQVTVSS 120 LKPEDTAVYVCNA GYDY 11A111/1- RFTISRDNAKNSVYLQMN 1667 RRSGYYSLS 1942 WGQGTQVTVSS 126 SLKPEDTAIYYCAA TSPHQYAY 13G71/1- RFTISRDNAKNTVYLQMN 1668 RVNYAAYSR 1943 WGQGTQVTVSS 124 SLKPEDTAVYYCGA LEHDYHY 13G74/1- RFTISRDNAKNTVYLQMN 1669 RVNYAAYSR 1944 WGQGTQVTVSS 125 SLKPEDTAVYYCGA LEHDYHY 11A71A/ RFTIARDDAKNTMYLQMN 1670 NRAGIYEY 1945 WGQGTQVTVSS 1-116 GMKPEDTAVYYCKM 22B101/1- RFDISRDNAKNTVYLQMN 1671 RLFMATPNQ 1946 WGQGTQVTVSS 123 RLKPEDTAVYYCAA GQYYY 11B42/1- RFAISRDNALNTVYLQMNS 1672 RPSYSTNNV 1947 WGQGTQVTVSS 123 LKPEDTAVYYCAA KSYRY 13E111/1- RFTISRDNGKNTVDLRMN 1673 RPAVVHTRK 1948 WGQGTQVTVSS 124 SLKPEDTAVYYCAA ESYPY 14H12/1- RFTISRDNAKNTVYLQMN 1674 RINYSVLTTT 1949 WGQGTQVTVSS 125 SLKPEDTAVYYCAA SSSYHY 13G101/ RFTISRDDAKNTVYLQMN 1675 DFRSTWLSS 1950 WGQGTQVTVSS 1-123 SLKAEDTAIYYCAA SGSSYTY 13G41/1- RFTVSRDNAQNTVYLQMN 1676 RRIVNVEGA 1951 WGQGTQVTVSS 121 SLKIEDTGVYYCNA YRDY 22B910/1- RFTISGDDARNTVYLQMN 1677 KVVVAVEGA 1952 WGQGTQVTVSS 121 SLNPEDTAVYYCNA KYDY 21A81/1- RFTISRDNSENTVYFEMG 1678 CRISWCAGA 1953 WGQGTQVTVSS 122 SLKPEDTAVYYCAI ESDYGY 21A92/1- RFTISRDNAKGTVYLQMS 1679 DSITDRRSV 1954 WGQGTQVTVSS 127 SLKPEDTAVYYCAA AVAHTSYYY 22C712/1- RFTISRDNTKNTVYLQMNS 1680 RLSSVAVAS 1955 WGQGTQVTVSS 123 MRPEDTAVYYCAA TRYDY 11A13/1- RFTISRDIANDVVYLQMNS 1681 NRQSGVAS 1956 WGQGTQVTVSS 125 LEPEDTAVYYCAR ENLRLYTY 13G93/1- RFTISRDNAKNTLYLQTTS 1682 DKQSGVSV 1957 WGQGTQVTVSS 123 LRPEDTGVYYCAE NPKYAY 12C52/1- RFTISKDNAKNTVYLQMNS 1683 QTLGRSLYD 1958 WGQGTQVTVSS 118 LKPEDTAVYYCAA Y 12C61/1- RFTISRDNAKNTVYLQMN 1684 SRYSGGVA 1959 WGQGTQVTVSS 126 SLKPEDTAVYYCAA TARRSEYHY 21A61/1- RFTISRDYAKNTWYLQMN 1685 VATYSRNVG 1960 WGQGTQVTVSS 125 TLKPEDTAAYYCAA SVRNYDY 11A121/1- RFTISRDNAKNTVQLQMN 1686 SQPLNYYTY 1961 WGQGTQVTVSS 126 SLKPEDTAVYYCAV YDARRYDY 11A91/1- RFTISRDNAKNTVYLQMTS 1687 TPVYYQRYY 1962 WGQGTQVTVSS 124 LMPEDTAVYYCAD DQNAYDY 13G72/1- RFTISRDNAKNTVYLQMN 1688 KYYSYYAYD 1963 WGQGTQVTVSS 118 SLKPEDTAVYYCAA Y 13E81/1- RFTISRDNAKDTMYLQMN 1689 SPVYYIDYS 1964 WGQGTQVTVSS 124 VLKPEDTAVYYCAA SQYKYGY 11B31/1- RFTISRDNAKNTVYLRMNS 1690 QIYYRTNYY 1965 WGQGTQVTVSS 124 LKPEDTAVYYCAT SQNAYDY 13G81/1- RFTISRDDAKNTVYLQMN 1691 QIYYRTNYY 1966 WGQGTQVTVSS 124 SLKPEDTAVYLCAA SQNNYDY 21A53/1- RVTISRDDAKNTVYLRMN 1692 QIYYRTNYY 1967 WGQGTQVTVSS 124 SLKPEDTAVYYCAA SQNVYDY 14H51/1- RFTISRDDAKNTVYLRMNS 1693 QIYYRTNYY 1968 WGQGTQVTVSS 124 LKPEDTAVYYCAA SQNEYDY 21A21/1- RFTISRDNAKNTVYLRMNS 1694 QIYYRTNYS 1969 WGQGTQVTVSS 124 LKPEDTAVYYCAA SQSNYDY 21A111/1- RFTIARDDAKNTVYLQMN 1695 ATAYRTNYS 1970 WGQGTQVTVSS 124 SLKPEDTAVYYCAL SRDKYDY 22B1212/ RFTISRDNAKNTLYLQMNS 1696 YLSFYSDYE 1971 WGQGTQVTVSS 1-122 LKPEDTAVYYCAK VYDY 11A31/1- RFTISRDNAKNGVSLQMD 1697 DPTYGSGR 1972 WGQGTQVTVSS 120 SLKAEDTAVYYCAS WTY 13E51/1- RFTISSDNAKNTVYLQMNS 1698 SVTYYSGSH 1973 WGQGTQVTVSS 128 LKPEDTALYYCAR AYTQEGGY AR 12D121/1- RFTISRDDAKNTVYLQMN 1699 RQPYASGS 1974 WGQGTQVTVSS 126 SLKPEDTAVYYCAA HYSSTQYTY 13F121/1- RFTISIDNAKNTVYLQMNN 1700 LYRGRSVYD 1975 WGQGTQVTVSS 119 LTPEDTAVYYCAA D 13G121/ RFTISRDNAKNTVYLQMD 1701 SSYGSTYYS 1976 WGQGTQVTVSS 1-127 SLKPEDTAVYYCAA QGRAYYYD Y 22B41/1- RFTISKENAKNTVYLQMTIL 1702 SPYGPLYRS 1977 WGQGTQVTVSS 124 KPEDTAVYYCAS THYYDY 12D71/1- RFTISRDSAKDTVYLQMN 1703 TTYYSGSYI 1978 WGQGTQVTVSS 125 NLKPEDTAVYYCAA STLSTSYNY 13F42/1- RFTISRDNAKNAVNLQMS 1704 GSY 1979 RGQGTQVTVSS 111 NLKPEDTALHYCTI 12C101/1- RFTISRDNARNAVNPQMN 1705 GSY 1980 RGQGTQVTVSS 111 NLKPEDTAVYYCTI 14H91/1- RFTISRDNAKNAVYLQMN 1706 RDSSTLDST 1981 WGRGTQVTVSS 127 SLKPEDTAVYYCAA YYVGGSYN Y 13F41/1- RFTISRDNAKNAVNLQMS 1707 GSY 1982 RGQGTQVTVSS 111 NLKPEDTALYYCTI 14H21/1- RFTISRSIAENTVYLQMNK 1708 RMYGSDWL 1983 WGQGTQVTVSS 125 VKPEDTAVYYCAA PRPEDFDS 22B610/1- RFTISRDNAKNTVYLQMN 1709 DVSPSYGS 1984 WGQGTQVTVSS 120 SLKPEDTAVYYCNA RWYG 12C32/1- RFTISRDNAKSTMYLQMN 1710 RREYSTIYT 1985 WGQGTQVTVSS 127 SLKPEDTAVYYCAA ARYPGEYVY 12D61/1- RFTISRDNAKNTLYLQMNS 1711 RERGIYDS 1986 WGQGTQVTVSS 116 LKAEDTAVYYCNA 13G31/1- RFTISRDDAKNSIYLQMNT 1712 DDYLGGDN 1987 WGQGTQVTVSS 125 LKPEDTAVYYCAA WYLGPYDS 22C65/1- RFTISRDNAKNTVYLQMN 1713 RKTYRSLTY 1988 WGQGTQVTVSS 124 SLIPEDTAVYYCAA YGEYDS 11A71/1- RFTVSRDNGRNTVYLLQMN 1714 DRLLYYSSG 1989 WGQGTQVTVSS 125 SLKPEDTAVYYCNV YYQTSVDV 11B91/1- RFTISRDKAKNTVLLQMDS 1715 DRFLYYSAG 1990 WGQGTQVTVSS 125 LKPEDTAVYYCNA RYDTGSDI 11A81/1- RFTISRDKAKNALYLQMNS 1716 DRVLYYSDS 1991 WGQGTQVTVSS 125 LKPEDMAVYYCNA RYYTGSNY 11B121/1- RFTISRDNAKNTVYLEMNN 1717 DRALYRNYS 1992 WGQGTQVTVSS 127 LKPEDTAVYYCNA DGRYYTGY DY 12D31/1- RFTISRDNAENTVYLQMN 1718 RILSRNY 1993 WGQGNQVTVSS 115 NLKPEDMAVYYCNA 11B51/1- RFTISRDNAKSTVYLQMDS 1719 SRRFYSGLY 1994 WGQGTQVTVSS 127 LKPEDTAVYYCAA YYTDDAYEY 13G51/1- RFTISRDNSKNTVSLQMN 1720 SRRFYSGLV 1995 WGQGTQVTVSS 127 SLKPEDTAVYYCAA YYSVDAYEN 13F82A/1- RFTIFRDNAENTAYLQMNS 1721 TKRYYSIKY 1996 WGQGTQVTVSS 130 LNPEDTAVYYCAV YSTVEDYEY 13E101/1- RFTISRDNAESTVYLQMNS 1722 NRYYCSTY 1997 WGQGTQVTVSS 128 LKAEDTAVYYCAA GCLSTPRQY DY 22B85/1- RFAISRDGAKNTVYLQMD 1723 RVYTGTYG 1998 WGQGTQVTVSS 120 SLKPEDTAVYYCNL GRNY 11B12/1- RFAISRDNAKNTVYLQMH 1724 GYSLPAFDS 1999 WGPGTQVTVSS 118 SLKPEDTAVYHCAT 13G61/1- RFTISRDNADNTVTLQMN 1725 GIDLYTFHY 2000 FGQGTQVTVSS 118 SLKPEDTAVYYCAG 14H41/1- RFTISRDNADNTVTLQMN 1726 GIDLYTFDY 2001 FGQGTQVTVSS 118 SLKPEDTAVYYCAG 11B81/1- RFTISRDNAEDLLYLQMNL 1727 NSDSGFDS 2002 WGQGTQVTVSS 126 LKPEDTAVYYCAA YSVWAAYEY 11C11/1- RFTISKNTAENTVYLQMNS 1728 LLTVWDTYKY 2003 WGQGTQVTVSS 121 LKPEDTAVYYCAV 12D92/1- RFTISRANDNNPLYLQMNT 1729 TNRWYTGV 2004 WGQGTQVTVSS 123 LKPEDTAVYYCAA YDLPSRYEY 13E61/1- RFTISRDNAINSLYLQMDT 1730 TNRWFSAV 2005 WGQGTQVTVSS 123 LKPEDTAVYYCAA YDLPSRYTY 22B71/1- RFSISRDNALKTVYLQMNS 1731 DLPSRL 2006 WGQGTQVTVSS 114 LKPEDTAVYYCYA 21A121/1- RFTISRDYDNSPVYLQMN 1732 TSRWYSAV 2007 WGQGTQVTVSS 123 TLKPEDTAVYYCAA YDLPTRYDY 13F101/1- RFTISRDNAKNTVYLQMN 1733 DRSYSIDYR 2008 WGQGTQVTVSS 124 SLEREDTAVYYCAA HPDSYSY 11A43/1- RFTISRDNAKNTVTLQMNS 1734 DPLLFYGVG 2009 WGQGTQVTVSS 123 LRPEDTAVYYCAA SADVDY 12C81/1- RFTISRDNAKNSVYLQMN 1735 ISGLVQRDY 2010 WGQGTQVTVSS 117 SLKPEDTSVYYCNV 11B21/1- RFTISRDNARNIVYLQMN 1736 GGHLLGYD 2011 WGQGTQVTVSS 124 SLKPEDTAVYYCAA VQWEPDY 11B71/1- RFTISRDNAKNIVYLQMN 1737 HKRTYELGA 2012 WGQGTQVTVSS 126 SLKPEDTAVYYCAA HSTDFGS 12C121/1- RFTISRDNAKNAVYLQMN 1738 AVFVDSGDF 2013 WGQGTQVTVSS 126 SLKPEDTGVYYCAT SVCRGVGY 22C51/1- RFTISRDNAKNTVYLQMN 1739 LRSWPRGV 2014 WGQGTQVTVSS 121 SLKPEDTAVYYCAA DSGS 12D11/1- RFTISRDNTKNTIDLQINSL 1740 DVIYKNIGS 2015 WGQGTQVTVSS 123 KPEDTAVYYCAA GSFDY 12D14/1- RFTISRDNTKNTIDLQINSL 1741 DVIYKNIGS 2016 WGQGTQVTVSS 123 KPEDTAVYYCAA GSFDY 12C111/1- RFIVSRDNAKNTADLQMN 1742 SKWYGGFG 2017 WGQGTQVTVSS 123 DLKPEDTAVYYCGA DTDIEY 22B55/1- RFIVSRDNAKNTADLQMN 1743 SKWYGGFG 2018 WGQGTQVTVSS 123 ELKPEDTAVYYCGA DTDIEY 14H121/1- RFTISRDDAKNTVFLQMNS 1744 DGPFGN 2019 WGQGTQVTVSS 113 LKPEDTAVYYCNV 12C71/1- RFTISRENVKDTMYLQMN 1745 SRHSGNTLS 2020 WGQGTQVTVSS 125 SLQAEDTGVYYCVR FSLKYDY 21A31/1- RFTISRDNARNTAYLQMD 1746 RRWSGTLS 2021 WGQGTQVTVSS 125 SLRPEDTAVYSCAA LFDNEYYY 12C91/1- RFTISRGNAKNTVNLQMN 1747 DNYRDSYLE 2022 WGQGTQVTVSS 121 SLKPEDTAVYYCAA YDY 14H81/1- RFTISRDNTESTGYLQMN 1748 SWDHGDYV 2023 WGQGTQVTVSS 125 NLKPEDTAVYYCAA DGGFFYDY 12C42/1- RFTISRDNAKNMVYLQMN 1749 AGSAGPPSI 2024 WGQGTQVTVSS 124 SLKPEDTAVYYCAA DRQYDY 12D102/1- RFTISRDYAKNTAYLQMNS 1750 DVRDSIWRSY 2025 WGQGTQVTVSS 118 LKPEDTAVYYCVA 11A52/1- RFTISRDNAENTVYLQMN 1751 GWGATQAQ 2026 WGQGTQVTVSS 120 SLTPEDTAVYYCAS SGF 14H111/1- RFTISRDNAENTVYLLMNS 1752 GWGATQAQ 2027 WGQGTQVTVSS 120 LIPEDTAVYYCAT HGF 11B61/1- RFTIYRDNAQNTMYLQMN 1753 SSSLATISQ 2028 WGQGTQVTVSS 120 SLKPEDTAVYYCAA PSS 12E42/1- RFTVSRDNAKNTVYLQMN 1754 KAVTSRDHEY 2029 WGQGTQVTVSS 118 SLKPEDTAVYYCNA 13F81A/1- RSTISRENAENTVYLQMN 1755 NSDEFYSGT 2030 WGQGTQVTVSS 128 SLKPEDTAVYYCAA LKLQSRMVEY 11B102/1- RFTISRDNAKKTAYLLIMNS 1756 GWKTDEYVK 2031 WGQGTQVTVSS 118 LRPEDTAVYYCVG 21A41/1- RFTISRDNTKNTVYLQMNS 1757 GWVTPSYE 2032 WGQGTQVTVSS 120 LKPEDTAVYYCRP YGN 14H101/1- RFTISRDNAKNTVDLQMS 1758 DQKYGMSY 2033 WGQGTQVTVSS 128 SLKPEDTAAYYCAA SRLWLVSEY EY 12E21/1- RFTISRDNAENLVYLQMN 1759 GWGDSAY 2034 WGQGTQVTVSS 115 NLKPEDTAVYYCSA 13F21/1- RFTISRDAAANLVYLQMNS 1760 NGIESYGW 2035 WTVGTQVTVSS 123 LKPSDTAIYSCNA GNRHFNY 12E33/1- RFIISRDNAKNTVYLQINSL 1761 GGWGTGRY 2036 WGVGTQVTVSS 119 KPEDTAVYYCAA NY 13G11/1- RFTISRDNAKNTVYLQMN 1762 GWGTAPLS 2037 WGQGTQVTVSS 122 NLKSEDTAVYYCAP TSVY 118N121_A6_2_OK/ RFTFSRDNAKNTVYLQMN 1763 DRFNTIANL 2038 WGQGTQVTVSS 1-123 SLKPEDTAIYLCAV PGEYDY 118N121_B8_1_OK/ RFTISRDNAKNTVYLQMN 1764 ETFNSISNLP 2039 WGQGTQVTVSS 1-135 SLKPEDTAVYYCAC GEYDY 118N121_A2_2_OK/ RFTIYRDNAKNWYLQMS 1765 DHFTFMSNL 2040 WGQGTQVTVSS 1-124 VLNGEDTAVYYCAA PSEYDY 118N121_A8_2_OK/ RFTISRDNAKNTVYLQMN 1766 RKIYRSLSY 2041 WGQGTQVTVSS 1-124 SLKPEDTAVYYCAA YGDYDS 118N121_B3_1_OK/ RFTISRDEAKNTVYLEMNS 1767 RLTMATPNQ 2042 WGQGTQVTVSS 1-123 LKTDDTAVYYCAA SQYYY 118N121_A5_2_OK/ RFTISRDNAKTIVYLQMNS 1768 SGIGTDN 2043 WGQGIEVTVSS 1-114 LQPEDTARYYCRY 118N121_A9_2_OK/ RFTISRDNAKTTVYLQMNS 1769 SGIGTDN 2044 WGQGIEVTVSS 1-114 LQPEDTARYYCRY 118N121_A7_1_OK/ RFTISRDNAKNTVYLQMN 1770 DILYKTDIYY 2045 WGQGTQVTVSS 1-122 SLKPEDTAVYYCYA RNDF 118N121_A10_1_OK/ RLPISSDNAKKTVYLQMDS 1771 LPYTICPVVV 2046 WGKGTQVTVSS 1-131 LKPEDTAVYYCAA KKGAVYYG VDDY 118N121_A11_1_OK/ RFTISRDNDENMLYLQMN 1772 GFSDRSFAV 2047 KGQGTQVTVSS 1-120 SLKPEDTAVYYCAT TH 118N121_B7_4_OK/ RFTISRDNAKNTVFLQMNS 1773 DFRLARLRV 2048 WGQGTQVTVSS 1-124 LKPEDTAIYYCAA ADDYDY 118N121_B2_1_OK/ RFATSSDSAKNTVYLQMH 1774 LRTDYSINW 2049 WGQGTQVTVSS 1-130 SLKPEDTAVYYCAT ANCQRDSL YGY 118N121_B7_1_OK/ RFIISRDDSKNTVDLQMNS 1775 GGWTRTHP 2050 WGQGTQVTVSS 1-119 LKPEDTAVYYCNL FDY

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; or from the group of CDR 1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% “sequence identity” (as defined herein) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 “amino acid difference(s)” (as defined herein) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

In this context, by “suitably chosen” is meant that, as applicable, a CDR1 sequence is chosen from suitable CDR1 sequences (i.e. as defined herein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as defined herein), respectively. More in particular, the CDR sequences are preferably chosen such that the Nanobodies of the invention bind to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-1; and/or from the group consisting of the CDR3 sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR3 sequences listed in Table A-1.

Preferably, in the Nanobodies of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 “amino acid difference(s)” with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-1, respectively; and at least one of the CDR1 and CDR2 sequences present is suitably chosen from the group consisting of the CDR1 and CDR2 sequences, respectively, listed in Table A-1 or from the group of CDR1 and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1 and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1.

Most preferably, in the Nanobodies of the invention, all three CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1 or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least one of the CDR1, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1. Preferably, in this aspect, at least one or preferably both of the other two CDR sequences present are suitably chosen from CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences, respectively, listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 listed in Table A-1. Preferably, in this aspect, at least one and preferably both of the CDR1 and CDR2 sequences present are suitably chosen from the groups of CDR1 and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR1 and CDR2 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1 and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1, and either the CDR1 sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDR1 and CDR2 sequences, respectively, listed in Table A-1. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the corresponding CDR sequences listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, all three CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

Also, generally, the combinations of CDR's listed in Table A-1 (i.e. those mentioned on the same line in Table A-1) are preferred. Thus, it is generally preferred that, when a CDR in a Nanobody of the invention is a CDR sequence mentioned in Table A-1 or is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with a CDR sequence listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with a CDR sequence listed in Table A-1, that at least one and preferably both of the other CDR's are suitably chosen from the CDR sequences that belong to the same combination in Table A-1 (i.e. mentioned on the same line in Table A-1) or are suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR sequence(s) belonging to the same combination and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the CDR sequence(s) belonging to the same combination. The other preferences indicated in the above paragraphs also apply to the combinations of CDR's mentioned in Table A-1.

Thus, by means of non-limiting examples, a Nanobody of the invention can for example comprise a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1, a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination); and a CDR3 sequence that has more than 80% sequence identity with one of the CDR3 sequences mentioned in Table A-1 (but belonging to a different combination); or (2) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence, and one of the CDR3 sequences listed in Table A-1; or (3) a CDR1 sequence; a CDR2 sequence that has more than 80% sequence identity with one of the CDR2 sequence listed in Table A-1; and a CDR3 sequence that has 3, 2 or 1 amino acid differences with the CDR3 sequence mentioned in Table A-1 that belongs to the same combination as the CDR2 sequence.

Some particularly preferred Nanobodies of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence mentioned in Table A-1 that belongs to the same combination; (2) a CDR1 sequence; a CDR 2 listed in Table A-1 and a CDR3 sequence listed in Table A-1 (in which the CDR2 sequence and CDR3 sequence may belong to different combinations).

Some even more preferred Nanobodies of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; the CDR2 sequence listed in Table A-1 that belongs to the same combination; and a CDR3 sequence mentioned in Table A-1 that belongs to a different combination; or (2) a CDR1 sequence mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid differences with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence listed in Table A-1 that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for example comprise a CDR1 sequence mentioned in Table A-1, a CDR2 sequence that has more than 80% sequence identity with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and the CDR3 sequence mentioned in Table A-1 that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDR1, CDR2 and CDR3 sequences present are suitably chosen from one of the combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

According to another preferred, but non-limiting aspect of the invention (a) CDR1 has a length of between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, such as 16 and 17 amino acid residues: and/or (c) CDR3 has a length of between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates to a Nanobody in which the CDR sequences (as defined herein) have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 2051-2325.

Generally, Nanobodies with the above CDR sequences may be as further described herein, and preferably have framework sequences that are also as further described herein. Thus, for example and as mentioned herein, such Nanobodies may be naturally occurring Nanobodies (from any suitable species), naturally occurring V_(HH) sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences or Nanobodies, including but not limited to partially humanized Nanobodies or V₁ sequences, fully humanized Nanobodies or V_(HH) sequences, camelized heavy chain variable domain sequences, as well as Nanobodies that have been obtained by the techniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates to a humanized Nanobody, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR) to CDR3 respectively), in which CDR1 to CDR3 are as defined herein and in which said humanized Nanobody comprises at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).

In another preferred, but non-limiting aspect, the invention relates to a Nanobody in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 2051-2325. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody and one or more of the sequences of SEQ ID NO's: 2051-2325, in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies can be as further described herein.

In another preferred, but non-limiting aspect, the invention relates to a Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 2051-2325 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 2051-2325.

Another preferred, but non-limiting aspect of the invention relates to humanized variants of the Nanobodies of SEQ ID NO's: 2051-2325, that comprise, compared to the corresponding native V_(HH) sequence, at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).

The polypeptides of the invention comprise or essentially consist of at least one Nanobody of the invention. Some preferred, but non-limiting examples of polypeptides of the invention are given in SEQ ID NO's: 2051-2390.

It will be clear to the skilled person that the Nanobodies that are mentioned herein as “preferred” (or “more preferred”, “even more preferred”, etc.) are also preferred (or more preferred, or even more preferred, etc.) for use in the polypeptides described herein. Thus, polypeptides that comprise or essentially consist of one or more “preferred” Nanobodies of the invention will generally be preferred, and polypeptides that comprise or essentially consist of one or more “more preferred” Nanobodies of the invention will generally be more preferred, etc.

Generally, proteins or polypeptides that comprise or essentially consist of a single Nanobody (such as a single Nanobody of the invention) will be referred to herein as “monovalent” proteins or polypeptides or as “monovalent constructs”. Proteins and polypeptides that comprise or essentially consist of two or more Nanobodies (such as at least two Nanobodies of the invention or at least one Nanobody of the invention and at least one other Nanobody) will be referred to herein as “multivalent” proteins or polypeptides or as “multivalent constructs”, and these may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention. Some non-limiting examples of such multivalent constructs will become clear from the further description herein.

According to one specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least two Nanobodies of the invention, such as two or three Nanobodies of the invention. As further described herein, such multivalent constructs can provide certain advantages compared to a protein or polypeptide comprising or essentially consisting of a single Nanobody of the invention, such as a much improved avidity for HER2. Such multivalent constructs will be clear to the skilled person based on the disclosure herein: some preferred, but non-limiting examples of such multivalent Nanobody constructs are the constructs of SEQ ID NO's: 2326-2390.

According to another specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least one Nanobody of the invention and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is preferably also a Nanobody. Such proteins or polypeptides are also referred to herein as “multispecific” proteins or polypeptides or as “multispecific constructs”, and these may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention (as will become clear from the further discussion herein of some preferred, but non-limiting multispecific constructs). Such multispecific constructs will be clear to the skilled person based on the disclosure herein; some preferred, but non-limiting examples of such multispecific Nanobody constructs are the constructs of SEQ ID NO's: 2331-2390.

A multispecific polypeptide or protein comprising or essentially consists of at least one Nanobody of the invention and at least one other binding unit directed against another epitope or antigenic determinant on HER2 (which is preferably also a Nanobody) is also referred to as a “multiparatopic” protein or polypeptide or a “multiparatopic construct”.

Some preferred, but non-limiting examples of bivalent monospecific polypeptides of the invention are given in SEQ ID NO's: 2326-2330. Some preferred, but non-limiting examples of bispecific polypeptides of the invention are given in. SEQ ID NO's: 2331-2390. Some preferred, but non-limiting examples of biparatopic polypeptides of the invention are given in SEQ ID NO's: 2336-2390.

According to yet another specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least one Nanobody of the invention, optionally one or more further Nanobodies, and at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the Nanobody of the invention and/or to the resulting fusion protein. Again, such fusion proteins may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention. Some non-limiting examples of such amino acid sequences and of such fusion constructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, for example to provide a trivalent bispecific construct comprising two Nanobodies of the invention and one other Nanobody, and optionally one or more other amino acid sequences. Further non-limiting examples of such constructs, as well as some constructs that are particularly preferred within the context of the present invention, will become clear from the further description herein.

In the above constructs, the one or more Nanobodies and/or other amino acid sequences may be directly linked to each other and/or suitably linked to each other via one or more linker sequences. Some suitable but non-limiting examples of such linkers will become clear from the further description herein.

In one specific aspect of the invention, a Nanobody of the invention or a compound, construct or polypeptide of the invention comprising at least one Nanobody of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such Nanobodies, compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise Nanobodies sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention that comprise at least one Nanobody of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the Nanobody of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more Nanobodies of the invention are suitable linked to one or more serum proteins or fragments thereof (such as serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, Nanobodies or (single) domain antibodies that can bind to serum proteins such as serum albumin, serum immunoglobulins such as IgG, or transferrine); polypeptides in which a Nanobody of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more Nanobodies of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to WO 08/068,280 of Ablynx N.V.

Again, as will be clear to the skilled person, such Nanobodies, compounds, constructs or polypeptides may contain one or more additional groups, residues, moieties or binding units, such as one or more further amino acid sequences and in particular one or more additional Nanobodies (i.e. not directed against HER2), so as to provide a tri- or multispecific Nanobody construct. Some preferred, but non-limiting examples of bispecific polypeptides of the invention that bind serum albumin are given in SEQ ID NOs: 2331-2335.

Generally, the Nanobodies of the invention (or compounds, constructs or polypeptides comprising the same) with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the Nanobodies, compounds, constructs or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such Nanobodies, compound, constructs or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the invention comprises one or more (such as two or preferably one) Nanobodies of the invention linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that allow the resulting polypeptide of the invention to cross the blood brain barrier. In particular, said one or more amino acid sequences that allow the resulting polypeptides of the invention to cross the blood brain barrier may be one or more (such as two and preferably one) Nanobodies, such as the Nanobodies described in WO 02/057445, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In particular, polypeptides comprising one or more Nanobodies of the invention are preferably such that they:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to         10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²         moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²         moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to         10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles         or more and more preferably 10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about         10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,         more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as         between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;         and/or such that they:     -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)         and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a         t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶         s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as         between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence of the invention is preferably such that it will bind to HER2 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. In this respect, it will be clear to the skilled person that a polypeptide that contains two or more Nanobodies of the invention may bind to HER2 with an increased avidity, compared to a polypeptide that contains only one amino acid sequence of the invention.

Some preferred IC₅₀ values for binding of the amino acid sequences or polypeptides of the invention to HER2 will become clear from the further description and examples herein.

Other polypeptides according to this preferred aspect of the invention may for example be chosen from the group consisting of amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more “sequence identity” (as defined herein) with one or more of the amino acid sequences of SEQ ID NO's: 2326-2390, in which the Nanobodies comprised within said amino acid sequences are preferably as further defined herein.

Particularly preferred amino acid sequences of the invention (including but not limited to Nanobodies) and polypeptides of the invention (including polypeptides that comprise one or more Nanobodies of the invention) are preferably such that they bind to the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or such that they can compete with Herceptin® for binding to HER-2, and also:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to         10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²         moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²         moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to         10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles         or more and more preferably 10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about         10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,         more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as         between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;         and/or such that they:     -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)         and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a         t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶         s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as         between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Other particularly preferred amino acid sequences of the invention (including but not limited to Nanobodies) and polypeptides of the invention (including polypeptides that comprise one or more Nanobodies of the invention) are preferably such that they bind to the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or such that they can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2, and also:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to         10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²         moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²         moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to         10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles         or more and more preferably 10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about         10⁷ M⁻¹ s⁻¹ preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more         preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between         10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;         and/or such that they:     -   bind to HER2 with a k_(off) rate between s⁻¹ (t_(1/2)=0.69 s)         and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a         t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶         s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as         between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Some specifically particularly preferred polypeptides of the invention (including polypeptides that comprise one or more Nanobodies of the invention) are preferably such that they (i) bind to the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2, and (ii) bind to the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or can compete with Herceptin® for binding to HER-2, and (iii) also:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to         10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²         moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²         moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to         10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles         or more and more preferably 10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about         10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,         more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as         between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;         and/or such that they:     -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)         and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a         t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶         s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as         between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Another aspect of this invention relates to a nucleic acid that encodes an amino acid sequence of the invention (such as a Nanobody of the invention) or a polypeptide of the invention comprising the same. Again, as generally described herein for the nucleic acids of the invention, such a nucleic acid may be in the form of a genetic construct, as defined herein.

In another aspect, the invention relates to host or host cell that expresses or that is capable of expressing an amino acid sequence of the invention (such as a Nanobody) and/or a polypeptide of the invention comprising the same; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

Another aspect of the invention relates to a product or composition containing or comprising at least one amino acid of the invention (such as a Nanobody), at least one polypeptide of the invention and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

The invention further relates to methods for preparing or generating the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

The invention further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with HER2. Some preferred but non-limiting applications and uses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description hereinbelow.

Generally, it should be noted that the term Nanobody as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the Nanobodies of the invention can generally be obtained: (1) by isolating the V_(HH) domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring V_(HH) domain; (3) by “humanization” (as described herein) of a naturally occurring V_(HH) domain or by expression of a nucleic acid encoding a such humanized V_(HH) domain; (4) by “camelization” (as described herein) of a naturally occurring V_(H) domain from any animal species, and in particular a from species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized V_(H) domain; (5) by “camelisation” of a “domain antibody” or “Dab” as described by Ward et al (supra), or by expression of a nucleic acid encoding such a camelized V_(H) domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to the skilled person based on the disclosure herein and for example include the methods and techniques described in more detail herein.

One preferred class of Nanobodies corresponds to the V_(HH) domains of naturally occurring heavy chain antibodies directed against HER2. As further described herein, such V_(HH) sequences can generally be generated or obtained by suitably immunizing a species of Camelid with HER2 (i.e. so as to raise an immune response and/or heavy chain antibodies directed against HER2), by obtaining a suitable biological sample from said Camelid (such as a blood sample, serum sample or sample of B-cells), and by generating V_(HH) sequences directed against HER2, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person and/or are further described herein.

Alternatively, such naturally occurring V_(HH) domains against HER2, can be obtained from naïve libraries of Camelid V_(HH) sequences, for example by screening such a library using HER2, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naïve V_(HH) libraries may be used, such as V_(HH) libraries obtained from naïve V_(HH) libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.

Thus, in another aspect, the invention relates to a method for generating Nanobodies, that are directed against HER2. In one aspect, said method at least comprises the steps of:

-   a) providing a set, collection or library of Nanobody sequences; and -   b) screening said set, collection or library of Nanobody sequences     for Nanobody sequences that can bind to and/or have affinity for     HER2;     and -   c) isolating the amino Nanobody or Nanobodies that can bind to     and/or have affinity for HER2.

In such a method, the set, collection or library of Nanobody sequences may be a naïve set, collection or library of Nanobody sequences; a synthetic or semi-synthetic set, collection or library of Nanobody sequences; and/or a set, collection or library of Nanobody sequences that have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library of Nanobody sequences may be an immune set, collection or library of Nanobody sequences, and in particular an immune set, collection or library of V_(HH) sequences, that have been derived from a species of Camelid that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of Nanobody or V_(HH) sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) Nanobody sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating Nanobody sequences comprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species     of Camelid that express immunoglobulin sequences; -   b) screening said collection or sample of cells for (i) cells that     express an immunoglobulin sequence that can bind to and/or have     affinity for HER2; and (ii) cells that express heavy chain     antibodies, in which substeps (i) and (ii) can be performed     essentially as a single screening step or in any suitable order as     two separate screening steps, so as to provide at least one cell     that expresses a heavy chain antibody that can bind to and/or has     affinity for HER2;     and -   c) either (1) isolating from said cell the V_(HH) sequence present     in said heavy chain antibody; or (ii) isolating from said cell a     nucleic acid sequence that encodes the V_(HH) sequence present in     said heavy chain antibody, followed by expressing said V_(HH)     domain.

In the method according to this aspect, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a Camelid that has been suitably immunized with HER2 or a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820. Particular reference is made to the so-called “Nanoclone®” technique described in International application WO 06/079372 by Ablynx N.V.

In another aspect, the method for generating an amino acid sequence directed against HER2 may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences     encoding heavy chain antibodies or Nanobody sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode a heavy chain     antibody or a Nanobody sequence that can bind to and/or has affinity     for HER2;     and -   c) isolating said nucleic acid sequence, followed by expressing the     V_(HH) sequence present in said heavy chain antibody or by     expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acid sequences encoding heavy chain antibodies or Nanobody sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of heavy chain antibodies or V_(HH) sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of Nanobody sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of Nanobody sequences that have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences encoding heavy chain antibodies or V_(HH) sequences derived from a Camelid that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

Also encompassed within the present invention are methods for preparing and generating multiparatopic (such as e.g. biparatopic, triparatopic, etc.) amino acids of the invention.

Without being limiting, a method for preparing and generating biparatopic amino acids of the invention may comprise at least the steps of:

-   a) providing a nucleic acid sequence encoding an HER2 binding amino     acid sequence fused to a set, collection or library of nucleic acid     sequences encoding amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for an antigenic     determinant on HER2 different from the antigenic determinant     recognized by the HER2 binding amino acid sequence;     and -   c) isolating the nucleic acid sequence encoding an HER2 binding     amino acid sequence fused to the nucleic acid sequence obtained in     b), followed by expressing the encoded amino acid sequence.

The biparatopic amino acid sequence obtained in the method above, can subsequently be fused to one or more further sets, collections or libraries of nucleic acid sequences encoding amino acid sequences and again screened for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for an antigenic determinant on HER2 different from the antigenic determinant of the HER2 binding amino acid sequence and the antigenic determinant of b) in order to obtain a triparatopic or multiparatopic amino acid sequence respectively.

In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 binding amino acid sequence fused to the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

According to a particularly preferred aspect, a method for preparing and generating biparatopic amino acids of the invention may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences,     in which each nucleic acid sequence in said set, collection or     library encodes a fusion protein that comprises a first amino acid     sequence that can bind to and/or has affinity for a first antigenic     determinant, part, domain or epitope on HER2 that is fused     (optionally via a linker sequence) to a second amino acid sequence,     in which essentially each second amino acid sequence (or most of     these) is a different member of a set, collection or library of     different amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for a second antigenic     determinant, part, domain or epitope on HER2 different from the     first antigenic determinant, part, domain or epitope on HER-2;     and -   c) isolating the nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for a second antigenic     determinant, part, domain or epitope on HER2 different from the     first antigenic determinant, part, domain or epitope on HER-2,     obtained in b), optionally followed by expressing the encoded amino     acid sequence.

In this preferred method, the first amino acid sequence in the fusion protein encoded by said set collection or library of nucleic acid sequences may be the same amino acid sequence for all members of the set, collection or library of nucleic acid sequences encoding the fusion protein; or the first amino acid sequence in the fusion protein encoded by said set collection or library of nucleic acid sequences may also be a member of a set collection or library of different amino acid sequences.

Again, in such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 binding amino acid sequence fused to the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In step b), the set, collection or library of nucleic acid sequences may also be screened for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for both the first antigenic determinant, part, domain or epitope on HER2 and the second antigenic determinant, part, domain or epitope on HER2. This may for example be performed in a subsequent steps (i.e. by in a first step screening or selecting for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for the second antigenic determinant, part, domain or epitope on HER2, and subsequently in a second step selecting or screening for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for the first antigenic determinant, part, domain or epitope on HER2; or visa versa) or in a single step (i.e. by simultaneously screening or selecting for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for both the first antigenic determinant, part, domain or epitope on HER2 and the second antigenic determinant, part, domain or epitope on HER2).

In a preferred aspect of the above method, the first amino acid sequence used in step a) is preferably such that (i) it can bind to and/or has affinity for the Herceptin® binding site on HER2 (and may in particular be directed against domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or (ii) competes with Herceptin® for binding to HER-2; and in step b), the set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode (i) an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or (ii) an amino acid sequence that can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2.

Alternatively, the first amino acid sequence used in step a) is preferably such that (i) it can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or (ii) competes with Omnitarg for binding to HER-2; and in step b), the set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode (i) an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or (ii) an amino acid sequence that can compete with Herceptin® for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequences that encode) amino acid sequences that compete with Herceptin® or Omnitarg, respectively, may be performed using generally known methods for screening or selecting for competitors of known binding molecules, which may for example involve performing the screening or selection in the presence of the binding molecule and/or determining the binding affinity of the compound(s) to be screened in the presence of the binding molecule.

It is also possible, in step b), to screen for nucleic acid sequences that both (i) encode an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or that can compete with Omnitarg® (or the Omnitarg Fab used in Example 9) for binding to HER-2; and that also (ii) encode an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or that can compete with Herceptin® for binding to HER-2. Again, this may be performed in separate steps or a single step, and by selecting or screening in the presence of Herceptin® and/or Omnitarg, as applicable.

It will also be clear to the skilled person that the above methods may be performed by screening a set, collection or library of amino acid sequences that correspond to (e.g. are encoded by) the nucleic acid sequences used in the above method; and such methods form further aspects of the invention.

The invention in a further aspect provides a method for preparing and generating biparatopic amino acids of the invention which comprises at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences,     in which each nucleic acid sequence in said set, collection or     library encodes a fusion protein that comprises a first amino acid     sequence that can bind to and/or has affinity for a first antigenic     determinant, part, domain or epitope on HER2 that is fused via a     linker sequence to a second amino acid sequence that has can bind to     and/or has affinity for a second antigenic determinant, part, domain     or epitope on HER2 (which may be the same or different as the first     antigenic determinant, part, domain or epitope on HER2), in which     essentially each nucleic acid sequence (or most of these) encodes a     fusion protein with a different linker sequence so as to provide a     set, collection or library encoding different fusion proteins; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for the first and     second antigenic determinant, part, domain or epitope on HER2;     and -   c) isolating the nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for the first and     second antigenic determinant, part, domain or epitope on HER2,     optionally followed by expressing the encoded amino acid sequence.

As will be clear to the skilled person, this method can be used to screen for suitable or even optimal linker lengths for linking the first and second amino acid sequence. For example, in this aspect, the first amino acid sequence may be an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or that can compete with Omnitarg (or the Omnitarg Fab used in Example 9); and the second amino acid sequence may be an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or that can compete with Herceptin® for binding to HER-2 (or visa versa). The screening and selection step b) may be performed as further described above.

Another method for preparing and generating biparatopic amino acids of the invention may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences     encoding amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for a set, collection or library of nucleic acid sequences     that encode an amino acid sequence that can bind to and/or has     affinity for HER2; -   c) ligating said set, collection or library of nucleic acid     sequences that encode an amino acid sequence that can bind to and/or     has affinity for HER2 to another nucleic acid sequence that encodes     an amino acid sequence that can bind to and/or has affinity for HER2     (e.g. a nucleic acid sequence that encodes an amino acid sequence     that competes with Herceptin® for binding HER2);     and -   d) from the set, collection or library of nucleic acid sequences     obtained in c), isolating the nucleic acid sequences encoding a     biparatopic amino acid sequence that can bind to and/or has affinity     for HER2 (and e.g. further selecting for nucleic acid sequences that     encode a biparatopic amino acid sequence that antagonizes with     higher potency compared to the monovalent amino acid sequences),     followed by expressing the encoded amino acid sequence.

The biparatopic amino acid sequence obtained in the method above, can subsequently be fused to one or more further sets, collections or libraries of nucleic acid sequences encoding amino acid sequences that can bind to and/or have affinity for HER2 in order to obtain a triparatopic or multiparatopic amino acid sequence respectively.

In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

The set, collection or library of nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for HER2 can be obtained by any selection or screening method known in the art for the selection and/or screening of nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for HER2 and as, for example, described in the Examples section.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

Another method for preparing and generating biparatopic amino acids of the invention may comprise at least the steps of:

-   a) providing a first set, collection or library of nucleic acid     sequences encoding amino acid sequences; -   b) screening said first set, collection or library of nucleic acid     sequences for a nucleic acid sequence that encodes an amino acid     sequence that can bind to and/or has affinity for a first antigenic     determinant, part, domain or epitope on HER2; -   c) ligating the nucleic acid sequence encoding said amino acid     sequence that can bind to and/or has affinity for a first antigenic     determinant, part, domain or epitope on HER2 to another set,     collection or library of nucleic acid sequences encoding amino acid     sequences to obtain a set, collection or library of nucleic acid     sequences that encode fusion proteins; -   d) screening said set, collection or library of nucleic acid     sequences obtained in step c) for a nucleic acid sequence that     encodes an amino acid sequence that can bind a second antigenic     determinant, part, domain or epitope on HER2 different from the     first antigenic determinant, part, domain or epitope on HER-2;     and     -   e) isolating the nucleic acid sequence that encodes an amino         acid sequence that can bind to and/or has affinity for the first         and second antigenic determinant, part, domain or epitope on         HER2, optionally followed by expressing the encoded amino acid         sequence.

In a preferred aspect of the above method, the first amino acid sequence used in step a) is preferably such that (i) it can bind to and/or has affinity for the Herceptin® binding site on HER2 (and may in particular be directed against domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or (ii) competes with Herceptin® for binding to HER-2; and in step b), the set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode (i) an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or (ii) an amino acid sequence that can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2.

Alternatively, the first amino acid sequence used in step a) is preferably such that (i) it can bind to and/or has affinity for the Omnitarg binding site on HER2 (and may in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or (ii) competes with Omnitarg for binding to HER-2; and in step b), the set, collection or library of nucleic acid sequences is screened for nucleic acid sequences that encode (i) an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or (ii) an amino acid sequence that can compete with Herceptin® for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequences that encode) amino acid sequences that compete with Herceptin® or Omnitarg, respectively, may be performed using generally known methods for screening or selecting for competitors of known binding molecules, which may for example involve performing the screening or selection in the presence of the binding molecule and/or determining the binding affinity of the compound(s) to be screened in the presence of the binding molecule.

It is also possible, in step b), to screen for nucleic acid sequences that both (i) encode an amino acid sequence that can bind to and/or has affinity for the Omnitarg binding site on HER2 (and in particular domain II of HER2, more in particular the middle of domain II of HER2) and/or that can compete with Omnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2; and that also (ii) encode an amino acid sequence that can bind to and/or has affinity for the Herceptin® binding site on HER2 (and in particular domain IV of HER2, more in particular the C-terminus of domain IV of HER2) and/or that can compete with Herceptin® for binding to HER-2. Again, this may be performed in separate steps or a single step, and by selecting or screening in the presence of Herceptin® and/or Omnitarg, as applicable.

The biparatopic amino acid sequence obtained in the method above, can subsequently be fused to one or more further sets, collections or libraries of nucleic acid sequences encoding amino acid sequences that can bind to and/or have affinity for HER2 in order to obtain a triparatopic or multiparatopic amino acid sequence respectively.

In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

The set, collection or library of nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for HER2 can be obtained by any selection or screening method known in the art for the selection and/or screening of nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for HER2 and as, for example, described in the Examples section.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of the methods described herein can also be performed as a selection step. Accordingly the term “screening” as used in the present description can comprise selection, screening or any suitable combination of selection and/or screening techniques. Also, when a set, collection or library of sequences is used, it may contain any suitable number of sequences, such as 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collection or library of amino acid sequences may be obtained or defined by rational, or semi-empirical approaches such as computer modelling techniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two or more sequences that are variants from one another (e.g. with designed point mutations or with randomized positions), compromise multiple sequences derived from a diverse set of naturally diversified sequences (e.g. an immune library)), or any other source of diverse sequences (as described for example in Hoogenboom et al. (Nat Biotechnol 23:1105, 2005) and Binz et al. (Nat Biotechnol 2005, 23:1247)). Such set, collection or library of sequences can be displayed on the surface of a phage particle, a ribosome, a bacterium, a yeast cell, a mammalian cell, and linked to the nucleotide sequence encoding the amino acid sequence within these carriers. This makes such set, collection or library amenable to selection procedures to isolate the desired amino acid sequences of the invention. More generally, when a sequence is displayed on a suitable host or host cell, it is also possible (and customary) to first isolate from said host or host cell a nucleotide sequence that encodes the desired sequence, and then to obtain the desired sequence by suitably expressing said nucleotide sequence in a suitable host organism. Again, this can be performed in any suitable manner known per se, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences or Nanobody sequences directed against HER2 involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and/or heavy chain antibodies directed against HER2), obtaining a suitable biological sample from said transgenic mammal that contains (nucleic acid sequences encoding) said V_(HH) sequences or Nanobody sequences (such as a blood sample, serum sample or sample of B-cells), and then generating V_(HH) sequences directed against HER2, starting from said sample, using any suitable technique known per se (such as any of the methods described herein or a hybridoma technique). For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens et al. (Proc. Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5) can be used. For example, such heavy chain antibody expressing mice can express heavy chain antibodies with any suitable (single) variable domain, such as (single) variable domains from natural sources (e.g. human (single) variable domains, Camelid (single) variable domains or shark (single) variable domains), as well as for example synthetic or semi-synthetic (single) variable domains.

The invention also relates to the V_(HH) sequences or Nanobody sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said V_(HH) sequence or Nanobody sequence; and of expressing or synthesizing said V_(HH) sequence or Nanobody sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of the invention comprises Nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_(HH) domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V_(HH) sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a V_(H) domain from a conventional 4-chain antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art on humanization referred to herein. Again, it should be noted that such humanized Nanobodies of the invention can be obtained in any suitable mariner known per se (i.e. as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V_(HH) domain as a starting material.

Another particularly preferred class of Nanobodies of the invention comprises Nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_(H) domain, but that has been “camelized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V_(H) domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V_(HH) domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such “camelizing” substitutions are preferably inserted at amino acid positions that form and/or are present at the V_(H)-V_(L) interface, and/or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996), supra). Preferably, the V₁ sequence that is used as a starting material or starting point for generating or designing the camelized Nanobody is preferably a V_(H) sequence from a mammal, more preferably the V_(H) sequence of a human being, such as a V_(H)3 sequence. However, it should be noted that such camelized Nanobodies of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V_(H) domain as a starting material.

For example, again as further described herein, both “humanization” and “camelization” can be performed by providing a nucleotide sequence that encodes a naturally occurring V_(HH) domain or V_(H) domain, respectively, and then changing, in a manner known per se, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a “humanized” or “camelized” Nanobody of the invention, respectively. This nucleic acid can then be expressed in a manner known per se, so as to provide the desired Nanobody of the invention. Alternatively, based on the amino acid sequence of a naturally occurring V_(HH) domain or V_(H) domain, respectively, the amino acid sequence of the desired humanized or camelized Nanobody of the invention, respectively, can be designed and then synthesized de novo using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring V_(HH) domain or V_(H) domain, respectively, a nucleotide sequence encoding the desired humanized or camelized Nanobody of the invention, respectively, can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to provide the desired Nanobody of the invention.

Other suitable methods and techniques for obtaining the Nanobodies of the invention and/or nucleic acids encoding the same, starting from naturally occurring V_(H) sequences or preferably V_(HH) sequences, will be clear from the skilled person, and may for example comprise combining one or more parts of one or more naturally occurring V₁₄ sequences (such as one or more FR sequences and/or CDR sequences), one or more parts of one or more naturally occurring V_(HH) sequences (such as one or more FR sequences or CDR sequences), and/or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide a Nanobody of the invention or a nucleotide sequence or nucleic acid encoding the same (which may then be suitably expressed). Nucleotide sequences encoding framework sequences of V_(HH) sequences or Nanobodies will be clear to the skilled person based on the disclosure herein and/or the further prior art cited herein (and/or may alternatively be obtained by PCR starting from the nucleotide sequences obtained using the methods described herein) and may be suitably combined with nucleotide sequences that encode the desired CDR's (for example, by PCR assembly using overlapping primers), so as to provide a nucleic acid encoding a Nanobody of the invention.

As mentioned herein, Nanobodies may in particular be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences.

Thus, according to one preferred, but non-limiting aspect of the invention, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 108     according to the Kabat numbering is Q;     and/or: -   b) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 45     according to the Kabat numbering is a charged amino acid (as defined     herein) or a cysteine residue, and position 44 is preferably an E;     and/or: -   c) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 103     according to the Kabat numbering is chosen from the group consisting     of P, R and S, and is in particular chosen from the group consisting     of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which

-   a) the amino acid residue at position 108 according to the Kabat     numbering is Q;     and/or in which:

-   b) the amino acid residue at position 45 according to the Kabat     numbering is a charged amino acid or a cysteine and the amino acid     residue at position 44 according to the Kabat numbering is     preferably E;     and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S, and is     in particular chosen from the group consisting of R and S;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In particular, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 108     according to the Kabat numbering is Q;     and/or: -   b) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 44     according to the Kabat numbering is E and in which the amino acid     residue at position 45 according to the Kabat numbering is an R;     and/or: -   c) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 103     according to the Kabat numbering is chosen from the group consisting     of P, R and S, and is in particular chosen from the group consisting     of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which

-   a) the amino acid residue at position 108 according to the Kabat     numbering is Q;     and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat     numbering is E and in which the amino acid residue at position 45     according to the Kabat numbering is an R;     and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S, and is     in particular chosen from the group consisting of R and S;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In particular, a Nanobody against HER2 according to the invention may have the structure:

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which

-   a) the amino acid residue at position 108 according to the Kabat     numbering is Q;     and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat     numbering is E and in which the amino acid residue at position 45     according to the Kabat numbering is an R;     and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S, and is     in particular chosen from the group consisting of R and S;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In particular, according to one preferred, but non-limiting aspect of the invention, a Nanobody can generally be defined as a polypeptide comprising an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of A, G, E, D, G, Q,     R, S, L; and is preferably chosen from the group consisting of G, E     or Q; and -   a-2) the amino acid residue at position 45 according to the Kabat     numbering is chosen from the group consisting of L, R or C; and is     preferably chosen from the group consisting of L or R; and -   a-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of W, R or S; and is     preferably W or R, and is most preferably W; -   a-4) the amino acid residue at position 108 according to the Kabat     numbering is Q;     or in which: -   b-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of E and Q; and -   b-2) the amino acid residue at position 45 according to the Kabat     numbering is R; and -   b-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of W, R and S; and is     preferably W; -   b-4) the amino acid residue at position 108 according to the Kabat     numbering is chosen from the group consisting of Q and L; and is     preferably Q;     or in which: -   c-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of A, G, E, D, Q, R, S     and L; and is preferably chosen from the group consisting of G, E     and Q; and -   c-2) the amino acid residue at position 45 according to the Kabat     numbering is chosen from the group consisting of L, R and C; and is     preferably chosen from the group consisting of L and R; and -   c-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S; and is     in particular chosen from the group consisting of R and S; and -   c-4) the amino acid residue at position 108 according to the Kabat     numbering is chosen from the group consisting of Q and L; is     preferably Q;     and in which -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   a-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of A, G, E, D, G, Q,     R, S, L; and is preferably chosen from the group consisting of G, E     or Q;     and in which:

-   a-2) the amino acid residue at position 45 according to the Kabat     numbering is chosen from the group consisting of L, R or C; and is     preferably chosen from the group consisting of L or R;     and in which:

-   a-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of W, R or S; and is     preferably W or R, and is most preferably W;     and in which

-   a-4) the amino acid residue at position 108 according to the Kabat     numbering is Q;     and in which:

-   d) CDR', CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   b-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of E and Q;     and in which:

-   b-2) the amino acid residue at position 45 according to the Kabat     numbering is R;     and in which:

-   b-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of W, R and S; and is     preferably W;     and in which:

-   b-4) the amino acid residue at position 108 according to the Kabat     numbering is chosen from the group consisting of Q and L; and is     preferably Q;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   c-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of A, G, E, D, Q, R, S     and L; and is preferably chosen from the group consisting of G, E     and Q;     and in which:

-   c-2) the amino acid residue at position 45 according to the Kabat     numbering is chosen from the group consisting of L, R and C; and is     preferably chosen from the group consisting of L and R;     and in which:

-   c-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S; and is     in particular chosen from the group consisting of R and S;     and in which:

-   c-4) the amino acid residue at position 108 according to the Kabat     numbering is chosen from the group consisting of Q and L; is     preferably Q;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

Two particularly preferred, but non-limiting groups of the Nanobodies of the invention are those according to a) above; according to (a-1) to (a-4) above; according to b) above; according to (b-1) to (b-4) above; according to (c) above; and/or according to (c-1) to (c-4) above, in which either:

-   i) the amino acid residues at positions 44-47 according to the Kabat     numbering form the sequence GLEW (or a GLEW-like sequence as     described herein) and the amino acid residue at position 108 is Q;     or in which:

ii) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE (SEQ ID NO: 2448) or KQRE (SEQ ID NO: 2449) (or a KERE-like sequence as described) and the amino acid residue at position 108 is Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (SEQ ID NO: 2447) (or a GLEW-like sequence as defined herein) and the amino acid residue at position 108 is Q;

and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

i) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE (SEQ ID NO: 2448) or KQRE (SEQ ID NO: 2449) (or a KERE-like sequence) and the amino acid residue at position 108 is Q or L, and is preferably Q;

and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In the Nanobodies of the invention in which the amino acid residues at positions 43-46according to the Kabat numbering form the sequence KERE (SEQ ID NO: 2448) or KQRE (SEQ ID NO: 2449), the amino acid residue at position 37 is most preferably F. In the Nanobodies of the invention in which the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (SEQ ID NO: 2447), the amino acid residue at position 37 is chosen from the group consisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the amino acid residues present on the positions mentioned above, the Nanobodies of the invention can generally be classified on the basis of the following three groups:

i) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW (SEQ ID NO: 2447) at positions 44-47 according to the Kabat numbering and Q at position 108according to the Kabat numbering. As further described herein, Nanobodies within this group usually have a V at position 37, and can have a W, P, R or S at position 103, and preferably have a W at position 103. The GLEW group also comprises some GLEW-like sequences such as those mentioned in Table A-3 below. More generally, and without limitation, Nanobodies belonging to the GLEW-group can be defined as Nanobodies with a G at position 44 and/or with a W at position 47, in which position 46 is usually E and in which preferably position 45 is not a charged amino acid residue and not cysteine;

ii) The “KERE-group”: Nanobodies with the amino acid sequence KERE (SEQ ID NO: 2448) or

KQRE (SEQ ID NO: 2449) (or another KERE-like sequence) at positions 43-46 according to the Kabat numbering and Q or L at position 108 according to the Kabat numbering. As further described herein, Nanobodies within this group usually have a F at position 37, an L or F at position 47; and can have a W, P, R or S at position 103, and preferably have a W at position 103. More generally, and without limitation, Nanobodies belonging to the KERE-group can be defined as Nanobodies with a K, Q or R at position 44 (usually K) in which position 45 is a charged amino acid residue or cysteine, and position 47 is as further defined herein; iii) The “103 P, R, S-group”: Nanobodies with a P, R or S at position 103. These Nanobodies can have either the amino acid sequence GLEW (SEQ ID NO: 2447) at positions 44-47according to the Kabat numbering or the amino acid sequence KERE (SEQ ID NO: 2448) or KQRE (SEQ ID NO: 2449) at positions 43-46 according to the Kabat numbering, the latter most preferably in combination with an F at position 37 and an L or an F at position 47 (as defined for the KERE-group); and can have Q or L at position 108 according to the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. have characteristics of) two or more of these classes. For example, one specifically preferred group of Nanobodies has GLEW (SEQ ID NO: 2447) or a GLEW-like sequence at positions 44-47; P,R or S (and in particular R) at position 103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred to above describe and apply to Nanobodies in the form of a native (i.e. non-humanized) V_(HH) sequence, and that humanized variants of these Nanobodies may contain other amino acid residues than those indicated above (i.e. one or more humanizing substitutions as defined herein). For example, and without limitation, in some humanized Nanobodies of the GLEW-group or the 103 P, R, S-group, Q at position 108 may be humanized to 108L. As already mentioned herein, other humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V_(HH) sequence with the corresponding framework sequence of one or more closely related human V_(H) sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V_(HH) sequence (in any manner known per se, as further described herein) and the resulting humanized V_(HH) sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a Nanobody may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may be a Nanobody belonging to the GLEW-group (as defined herein), and in which CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of the invention may be a Nanobody belonging to the KERE-group (as defined herein), and CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may be a Nanobody belonging to the 103 P, R, S-group (as defined herein), and in which CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,S residues mentioned above, the Nanobodies of the invention can contain, at one or more positions that in a conventional V_(H) domain would form (part of) the V_(H)/V_(L) interface, one or more amino acid residues that are more highly charged than the amino acid residues that naturally occur at the same position(s) in the corresponding naturally occurring V_(H) sequence, and in particular one or more charged amino acid residues (as mentioned in Table A-2). Such substitutions include, but are not limited to, the GLEW-like sequences mentioned in Table A-3 below; as well as the substitutions that are described in the International Application WO 00/29004 for so-called “microbodies”, e.g. so as to obtain a Nanobody with Q at position 108 in combination with KLEW (SEQ ID NO: 2482) at positions 44-47. Other possible substitutions at these positions will be clear to the skilled person based upon the disclosure herein.

In one aspect of the Nanobodies of the invention, the amino acid residue at position 83 is chosen from the group consisting of L, M, S, V and W; and is preferably L.

Also, in one aspect of the Nanobodies of the invention, the amino acid residue at position 83 is chosen from the group consisting of R, K, N, E, G, I, T and Q; and is most preferably either K or E (for Nanobodies corresponding to naturally occurring V_(HH) domains) or R (for “humanized” Nanobodies, as described herein). The amino acid residue at position 84 is chosen from the group consisting of P, A, R, S, D T, and V in one aspect, and is most preferably P (for Nanobodies corresponding to naturally occurring V_(HH) domains) or R (for “humanized” Nanobodies, as described herein).

Furthermore, in one aspect of the Nanobodies of the invention, the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108, which in the Nanobodies are as mentioned above, will also be referred to herein as the “Hallmark Residues”. The Hallmark Residues and the amino acid residues at the corresponding positions of the most closely related human V_(H) domain, V_(H)3, are summarized in Table A-3.

Some especially preferred but non-limiting combinations of these Hallmark Residues as occur in naturally occurring V_(HH) domains are mentioned in Table A-4. For comparison, the corresponding amino acid residues of the human V_(H)3 called DP-47 have been indicated in italics.

TABLE A-3 Hallmark Residues in Nanobodies Position Human V_(H)3 Hallmark Residues  11 L, V; predominantly L L, M, S, V, W; preferably L  37 V, I, F; usually V F⁽¹⁾, Y, H, I, L or V, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G G⁽²⁾, E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾ or E⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R  83 R or K; usually R R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q or T; preferably K or R; most preferably K  84 A, T, D; P⁽⁵⁾, A, L, R, S, T, D, V; preferably P predominantly A 103 W W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W 104 G G or D; preferably G 108 L, M or T; Q, L⁽⁷⁾ or R; preferably Q or L⁽⁷⁾ predominantly L Notes: ⁽¹⁾In particular, but not exclusively, in combination with KERE (SEQ ID NO: 2448) or KQRE (SEQ ID NO: 2449) at positions 43-46. ⁽²⁾Usually as GLEW (SEQ ID NO: 2447) at positions 44-47. ⁽³⁾Usually as KERE (SEQ ID NO: 2448) or KQRE (SEQ ID NO: 2449) at positions 43-46, e.g. as KEREL (SEQ ID NO: 2450), KEREF (SEQ ID NO: 2451), KQREL (SEQ ID NO: 2452), KQREF (SEQ ID NO: 2453) or KEREG (SEQ ID NO: 2454) at positions 43-47. Alternatively, also sequences such as TERE (SEQ ID NO: 2455) (for example TEREL (SEQ ID NO: 2456)), KECE (SEQ ID NO: 2457) (for example KECEL (SEQ ID NO: 2458) or KECER (SEQ ID NO: 2459)), RERE (SEQ ID NO: 2460) (for example REREG (SEQ ID NO: 2461)), QERE (SEQ ID NO: 2462) (for example QEREG (SEQ ID NO: 2463)), KGRE (SEQ ID NO: 2464) (for example KGREG (SEQ ID NO: 2465)), KDRE (SEQ ID NO: 2466) (for example KDREV (SEQ ID NO: 2467)) are possible. Some other possible, but less preferred sequences include for example DECKL (SEQ ID NO: 2468) and NVCEL (SEQ ID NO: 2469). ⁽⁴⁾With both GLEW (SEQ ID NO: 2447) at positions 44-47 and KERE (SEQ ID NO: 2448) or KQRE (SEQ ID NO: 2449) at positions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, in combination with GLEW (SEQ ID NO: 2447) at positions 44-47. ⁽⁷⁾With the proviso that when positions 44-47 are GLEW (SEQ ID NO: 2447), position 108 is always Q in (non-humanized) V_(HH) sequences that also contain a W at 103. ⁽⁸⁾The GLEW group also contains GLEW-like sequences at positions 44-47, such as for example GVEW (SEQ ID NO: 2470), EPEW (SEQ ID NO: 2471), GLER (SEQ ID NO: 2472), DQEW (SEQ ID NO: 2473), DLEW (SEQ ID NO: 2474), GIEW (SEQ ID NO: 2475), ELEW (SEQ ID NO: 2476), GPEW (SEQ ID NO: 2477), EWLP (SEQ ID NO: 2478), GPER (SEQ ID NO: 2479), GLER (SEQ ID NO: 2480) and ELEW (SEQ ID NO: 2481).

TABLE A-4 Some preferred but non-limiting combinations of Hallmark Residues in naturally occurring Nanobodies. For humanization of these combinations, reference is made to the specification. 11 37 44 45 47 83 84 103 104 108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R L K P W G Q L F E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q L F L R V K P Q G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group L V G L W K S W G Q M V G L W K P R G Q

In the Nanobodies, each amino acid residue at any other position than the Hallmark Residues can be any amino acid residue that naturally occurs at the corresponding position (according to the Kabat numbering) of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables A-5 to A-8 mention some non-limiting residues that can be present at each position (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4 of naturally occurring V_(HH) domains. For each position, the amino acid residue that most frequently occurs at each position of a naturally occurring V_(HH) domain (and which is the most preferred amino acid residue for said position in a Nanobody) is indicated in bold; and other preferred amino acid residues for each position have been underlined (note: the number of amino acid residues that are found at positions 26-30 of naturally occurring V_(HH) domains supports the hypothesis underlying the numbering by Chothia (supra) that the residues at these positions already form part of CDR1).

In Tables A-5-A-8, some of the non-limiting residues that can be present at each position of a human V_(H)3 domain have also been mentioned. Again, for each position, the amino acid residue that most frequently occurs at each position of a naturally occurring human V_(H)3 domain is indicated in bold; and other preferred amino acid residues have been underlined.

For reference only, Tables A-5-A-8 also contain data on the V_(HH) entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at each amino acid position for a representative sample of 1118 V_(HH) sequences (data kindly provided by David Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The values for the V_(HH) entropy and the V_(HH) variability provide a measure for the variability and degree of conservation of amino acid residues between the 1118 V_(HH) sequences analyzed: low values (i.e. <1, such as <0.5) indicate that an amino acid residue is highly conserved between the V_(HH) sequences (i.e. little variability). For example, the G at position 8 and the G at position 9 have values for the V_(HH) entropy of 0.1 and 0 respectively, indicating that these residues are highly conserved and have little variability (and in case of position 9 is G in all 1118 sequences analysed), whereas for residues that form part of the CDR's generally values of 1.5 or more are found (data not shown). Note that (1) the amino acid residues listed in the second column of Tables A-5-A-8 are based on a bigger sample than the 1118 V_(HH) sequences that were analysed for determining the V_(HH) entropy and V_(HH) variability referred to in the last two columns; and (2) the data represented below support the hypothesis that the amino acid residues at positions 27-30 and maybe even also at positions 93 and 94 already form part of the CDR's (although the invention is not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat is used). For a general explanation of sequence entropy, sequence variability and the methodology for determining the same, see Oliveira et al., PROTEINS: Structure, Function and Genetics, 52: 544-552 (2003).

TABLE A-5 Non-limiting examples of amino acid residues in FR1 (for the footnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 1 E, Q Q, A, E — — 2 V V 0.2 1 3 Q Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E, D, Q, A 0.8 4 7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R 0.3 2 11 Hallmark residue: L, M, S, V, W; preferably L 0.8 2 12 V, I V, A 0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1 5 15 G G 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1 1 19 R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A, F, T 0.2 3 22 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I, L, S, T, V 1 6 25 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V 0.7 7 27 F S, F, R, L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A, G, R, F, Y 1.7 11 29 F, V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G N, S, E, G, A, D, M, T 1.8 11

TABLE A-6 Non-limiting examples of amino acid residues in FR2 (for the footnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0.1 1 37 Hallmark residue: F⁽¹⁾, H, I, L, 1.1 6 Y or V, preferably F⁽¹⁾ or Y 38 R R 0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S, T P, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7 6 44 Hallmark residue: G⁽²⁾, E⁽³⁾, A, D, Q, 1.3 5 R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾ or E⁽³⁾. 45 Hallmark residue: L⁽²⁾, R⁽³⁾, C, I, L, 0.6 4 P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 46 E, V E, D, K, Q, V 0.4 2 47 Hallmark residue: W⁽²⁾, L⁽¹⁾ or 1.9 9 F⁽¹⁾, A, G, I, M, R, S, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L 0.4 3 49 S, A, G A, S, G, T, V 0.8 3

TABLE A-7 Non-limiting examples of amino acid residues in FR3 (for the footnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 66 R R 0.1 1 67 F F, L, V 0.1 1 68 T T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A, F, T 0.3 4 71 R R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V 0.5 4 73 N, D, G N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G, N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D, K, R, S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V, L, A, F, G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L, F, V 0.1 1 81 Q Q, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2 82a N, G N, D, G, H, S, T 0.8 4 82b S S, N, D, G, R, T 1 6 82c L L, P, V 0.1 2 83 Hallmark residue: R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, 0.9 7 Q or T; preferably K or R; most preferably K 84 Hallmark residue: P⁽⁵⁾, A, D, L, R, S, T, V; 0.7 6 preferably P 85 E, G E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 3 88 A A, G, S 0.3 2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 1 91 Y, H Y, D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N, G, H, K, N, R, S, T, V, Y 1.4 10 94 K, R, T A, V, C, F, G, I, K, L, R, S or T 1.6 9

TABLE A-8 Non-limiting examples of amino acid residues in FR4 (for the footnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 103 Hallmark residue: W⁽⁴⁾, P⁽⁶⁾, 0.4 2 R⁽⁶⁾, S; preferably W 104 Hallmark residue: G or D; 0.1 1 preferably G 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1 107 T T, A, I 0.3 2 108 Hallmark residue: Q, L⁽⁷⁾ or 0.4 3 R; preferably Q or L⁽⁷⁾ 109 V V 0.1 1 110 T T, I, A 0.2 1 111 V V, A, I 0.3 2 112 S S, F 0.3 1 113 S S, A, L, P, T 0.4 3

Thus, in another preferred, but not limiting aspect, a Nanobody of the invention can be defined as an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:

-   i) one or more of the amino acid residues at positions 11, 37, 44,     45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering     are chosen from the Hallmark residues mentioned in Table A-3;     and in which: -   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

In particular, a Nanobody of the invention can be an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which. CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:

-   i) (preferably) one or more of the amino acid residues at positions     11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat     numbering are chosen from the Hallmark residues mentioned in Table     A-3 (it being understood that V_(HH) sequences will contain one or     more Hallmark residues; and that partially humanized Nanobodies will     usually, and preferably, [still] contain one or more Hallmark     residues [although it is also within the scope of the invention to     provide—where suitable in accordance with the invention—partially     humanized Nanobodies in which all Hallmark residues, but not one or     more of the other amino acid residues, have been humanized]; and     that in fully humanized Nanobodies, where suitable in accordance     with the invention, all amino acid residues at the positions of the     Hallmark residues will be amino acid residues that occur in a human     V_(H)3 sequence. As will be clear to the skilled person based on the     disclosure herein that such V_(HH) sequences, such partially     humanized Nanobodies with at least one Hallmark residue, such     partially humanized Nanobodies without Hallmark residues and such     fully humanized Nanobodies all form aspects of this invention);     and in which: -   ii) said amino acid sequence has at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 1 to     22, in which for the purposes of determining the degree of amino     acid identity, the amino acid residues that form the CDR sequences     (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are     disregarded;     and in which: -   iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably     as defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

TABLE A-9 Representative amino acid sequences for Nanobodies of the KERE, GLEW and P, R, S 1.03 group. The CDR's are indicated with XXXX KERE sequence no. 1 SEQ ID NO: 1 EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNA KNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2 SEQ ID NO: 2 QVKLEESGGGLVQAGGSLRLSCVGSGRTESXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTA SNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3 SEQ ID NO: 3 AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWERQTPGREREFVAXXXXXRFTI SRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4 SEQ ID NO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTI SRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5 SEQ ID NO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTI SMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6 SEQ ID NO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFT ISSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7 SEQ ID NO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFT IARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8 SEQ ID NO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFT ISTDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9 SEQ ID NO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTI SGDNAKRAIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10 SEQ ID NO: 10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTI SRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 11 SEQ ID NO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTI ARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12 SEQ ID NO: 12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFT ISRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13 SEQ ID NO: 13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFT ISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14 SEQ ID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFT VSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15 SEQ ID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTI SRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16 SEQ ID NO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFT VSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1 SEQ ID NO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFT SRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2 SEQ ID NO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRF KISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3 SEQ ID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTI SRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 sequence no. 1 SEQ ID NO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 sequence no. 2 SEQ ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFT ISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103 sequence no. 3 SEQ ID NO: 22 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRF KISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS

In particular, a Nanobody of the invention of the KERE group can be an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which:

-   i) the amino acid residue at position 45 according to the Kabat     numbering is a charged amino acid (as defined herein) or a cysteine     residue, and position 44 is preferably an E;     and in which:

-   ii) FR1 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-10 Representative FW1 sequences for Nanobodies of the KERE-group. KERE FW1 sequence no. 1 SEQ ID NO: 23 QVQRVESGGGLVQAGGSLRLSCAASGRTSS KERE FW1 sequence no. 2 SEQ ID NO: 24 QVQLVESGGGLVQTGDSLSLSCSASGRTFS KERE FW1 sequence no. 3 SEQ ID NO: 25 QVKLEESGGGLVQAGDSLRLSCAATGRAFG KERE FW1 sequence no. 4 SEQ ID NO: 26 AVQLVESGGGLVQPGESLGLSCVASGRDFV KERE FW1 sequence no. 5 SEQ ID NO: 27 EVQLVESGGGLVQAGGSLRLSCEVLGRTAG KERE FW1 sequence no. 6 SEQ ID NO: 28 QVQLVESGGGWVQPGGSLRLSCAASETILS KERE FW1 sequence no. 7 SEQ ID NO: 29 QVQLVESGGGTVQPGGSLNLSCVASGNTFN KERE FW1 sequence no. 8 SEQ ID NO: 30 EVQLVESGGGLAQPGGSLQLSCSAPGFTLD KERE FW1 sequence no. 9 SEQ ID NO: 31 AQELEESGGGLVQAGGSLRLSCAASGRTFN and in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-11 Representative FW2 sequences for Nanobodies of the KERE-group. KERE FW2 SEQ ID NO: 41 WFRQAPGKEREFVA sequence no. 1 KERE FW2 SEQ ID NO: 42 WFRQTPGREREFVA sequence no. 2 KERE FW2 SEQ ID NO: 43 WYRQAPGKQREMVA sequence no. 3 KERE FW2 SEQ ID NO: 44 WYRQGPGKQRELVA sequence no. 4 KERE FW2 SEQ ID NO: 45 WIRQAPGKEREGVS sequence no. 5 KERE FW2 SEQ ID NO: 46 WFREAPGKEREGIS sequence no. 6 KERE FW2 SEQ ID NO: 47 WYRQAPGKERDLVA sequence no. 7 KERE FW2 SEQ ID NO: 48 WFRQAPGKQREEVS sequence no. 8 KERE FW2 SEQ ID NO: 49 WFRQPPGKVREFVG sequence no. 9 and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-12 Representative FW3 sequences for Nanobodies of the KERE-group. KERE FW3 SEQ ID NO: 50 RFTISRDNAKNTVYLQMNSL sequence no. 1 KPEDTAVYRCYF KERE FW3 SEQ ID NO: 51 RFAISRDNNKNTGYLQMNSL sequence no. 2 EPEDTAVYYCAA KERE FW3 SEQ ID NO: 52 RFTVARNNAKNTVNLEMNSL sequence no. 3 KPEDTAVYYCAA KERE FW3 SEQ ID NO: 53 RFTISRDIAKNTVDLLMNNL sequence no. 4 EPEDTAVYYCAA KERE FW3 SEQ ID NO: 54 RLTISRDNAVDTMYLQMNSL sequence no. 5 KPEDTAVYYCAA KERE FW3 SEQ ID NO: 55 RFTISRDNAKNTVYLQMDNV sequence no. 6 KPEDTAIYYCAA KERE FW3 SEQ ID NO: 56 RFTISKDSGKNTVYLQMTSL sequence no. 7 KPEDTAVYYCAT KERE FW3 SEQ ID NO: 57 RFTISRDSAKNMMYLQMNNL sequence no. 8 KPQDTAVYYCAA KERE FW3 SEQ ID NO: 58 RFTISRENDKSTVYLQLNSL sequence no. 9 KPEDTAVYYCAA KERE FW3 SEQ ID NO: 59 RFTISRDYAGNTAYLQMNSL sequence no. 10 KPEDTGVYYCAT and in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-13 Representative FW4 sequences for Nanobodies of the KERE-group. KERE FW4 sequence no. 1 SEQ ID NO: 60 WGQGTQVTVSS KERE FW4 sequence no. 2 SEQ ID NO: 61 WGKGTLVTVSS KERE FW4 sequence no. 3 SEQ ID NO: 62 RGQGTRVTVSS KERE FW4 sequence no. 4 SEQ ID NO: 63 WGLGTQVTISS and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

With regard to framework 1, it will be clear to the skilled person that, when an amino acid sequence as outlined above is generated by expression of a nucleotide sequence, the first four amino acid sequences (i.e. amino acid residues 1-4 according to the Kabat numbering) may often be determined by the primer(s) that have been used to generate said nucleic acid. Thus, for determining the degree of amino acid identity, the first four amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27 to 30 are according to the Kabat numbering considered to be part of the framework regions (and not the CDR's), it has been found by analysis of a database of more than 1000 V_(HH) sequences that the positions 27 to 30 have a variability (expressed in terms of V_(HH) entropy and V_(HH) variability—see Tables A-5 to A-8) that is much greater than the variability on positions 1 to 26. Because of this, for determining the degree of amino acid identity, the amino acid residues at positions 27 to 30 are preferably also disregarded.

In view of this, a Nanobody of the KERE class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

-   i) the amino acid residue at position 45 according to the Kabat     numbering is a charged amino acid (as defined herein) or a cysteine     residue, and position 44 is preferably an E;     and in which: -   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the     Kabat numbering, has at least 80% amino acid identity with at least     one of the following amino acid sequences:

TABLE A-14 Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the KERE-group. KERE FW1 SEQ ID NO: 32 VESGGGLVQPGGSLRLSCAASG sequence no. 10 KERE FW1 SEQ ID NO: 33 VDSGGGLVQAGDSLKLSCALTG sequence no. 11 KERE FW1 SEQ ID NO: 34 VDSGGGLVQAGDSLRLSCAASG sequence no. 12 KERE FW1 SEQ ID NO: 35 VDSGGGLVEAGGSLRLSCQVSE sequence no. 13 KERE FW1 SEQ ID NO: 36 QDSGGGSVQAGGSLKLSCAASG sequence no. 14 KERE FW1 SEQ ID NO: 37 VQSGGRLVQAGDSLRLSCAASE sequence no. 15 KERE FW1 SEQ ID NO: 38 VESGGTLVQSGDSLKLSCASST sequence no. 16 KERE FW1 SEQ ID NO: 39 MESGGDSVQSGGSLTLSCVASG sequence no. 17 KERE FW1 SEQ ID NO: 40 QASGGGLVQAGGSLRLSCSASV sequence no. 18 and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4     of Nanobodies of the KERE-class;     and in which: -   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

A Nanobody of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which

-   i) preferably, when the Nanobody of the GLEW-class is a     non-humanized Nanobody, the amino acid residue in position 108 is Q; -   ii) FR1 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-15 Representative FW1 sequences for Nanobodies of the GLEW-group. GLEW FW1 SEQ ID NO: 64 QVQLVESGGGLVQPGGSLRL sequence no. 1 SCAASGFTFS GLEW FW1 SEQ ID NO: 65 EVHLVESGGGLVRPGGSLRL sequence no. 2 SCAAFGFIFK GLEW FW1 SEQ ID NO: 66 QVKLEESGGGLAQPGGSLRL sequence no. 3 SCVASGFTFS GLEW FW1 SEQ ID NO: 67 EVQLVESGGGLVQPGGSLRL sequence no. 4 SCVCVSSGCT GLEW FW1 SEQ ID NO: 68 EVQLVESGGGLALPGGSLTL sequence no. 5 SCVFSGSTFS and in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-16 Representative FW2 sequences for Nanobodies of the GLEW-group. GLEW FW2 SEQ ID NO: 72 WVRQAPGKVLEWVS sequence no. 1 GLEW FW2 SEQ ID NO: 73 WVRRPPGKGLEWVS sequence no. 2 GLEW FW2 SEQ ID NO: 74 WVRQAPGMGLEWVS sequence no. 3 GLEW FW2 SEQ ID NO: 75 WVRQAPGKEPEWVS sequence no. 4 GLEW FW2 SEQ ID NO: 76 WVRQAPGKDQEWVS sequence no. 5 GLEW FW2 SEQ ID NO: 77 WVRQAPGKAEEWVS sequence no. 6 GLEW FW2 SEQ ID NO: 78 WVRQAPGKGLEWVA sequence no. 7 GLEW FW2 SEQ ID NO: 79 WVRQAPGRATEWVS sequence no. 8 and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-17 Representative FW3 sequences for Nanobodies of the GLEW-group. GLEW FW3 SEQ ID NO: 80 RFTISRDNAKNTLYLQMNSLK sequence no. 1 PEDTAVYYCVK GLEW FW3 SEQ ID NO: 81 RFTISRDNARNTLYLQMDSLIP sequence no. 2 EDTALYYCAR GLEW FW3 SEQ ID NO: 82 RFTSSRDNAKSTLYLQMNDLK sequence no. 3 PEDTALYYCAR GLEW FW3 SEQ ID NO: 83 RFIISRDNAKNTLYLQMNSLGP sequence no. 4 EDTAMYYCQR GLEW FW3 SEQ ID NO: 84 RFTASRDNAKNTLYLQMNSLKS sequence no. 5 EDTARYYCAR GLEW FW3 SEQ ID NO: 85 RFTISRDNAKNTLYLQMDDLQS sequence no. 6 EDTAMYYCGR and in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-18 Representative FW4 sequences for Nanobodies of the GLEW-group. GLEW FW4 sequence no. 1 SEQ ID NO: 86 GSQGTQVTVSS GLEW FW4 sequence no. 2 SEQ ID NO: 87 LRGGTQVTVSS GLEW FW4 sequence no. 3 SEQ ID NO: 88 RGQGTLVTVSS GLEW FW4 sequence no. 4 SEQ ID NO: 89 RSRGIQVTVSS GLEW FW4 sequence no. 5 SEQ ID NO: 90 WGKGTQVTVSS GLEW FW4 sequence no. 6 SEQ ID NO: 91 WGQGTQVTVSS and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

-   i) preferably, when the Nanobody of the GLEW-class is a     non-humanized Nanobody, the amino acid residue in position 108 is Q;     and in which: -   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the     Kabat numbering, has at least 80% amino acid identity with at least     one of the following amino acid sequences:

TABLE A-19 Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the KERE-group. GLEW FW1 SEQ ID NO: 69 VESGGGLVQPGGSLRLSCAASG sequence no. 6 GLEW FW1 SEQ ID NO: 70 EESGGGLAQPGGSLRLSCVASG sequence no. 7 GLEW FW1 SEQ ID NO: 71 VESGGGLALPGGSLTLSCVFSG sequence no. 8 and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4     of Nanobodies of the GLEW-class;     and in which: -   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein. In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which

-   i) the amino acid residue at position 103 according to the Kabat     numbering is different from W;     and in which: -   ii) preferably the amino acid residue at position 103 according to     the Kabat numbering is P, R or S, and more preferably R;     and in which: -   iii) FR1 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-20 Representative FW1 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 FW1 SEQ ID NO: 92 AVQLVESGGGLVQAGGSLRL sequence no. 1 SCAASGRTFS P, R, S 103 FW1 SEQ ID NO: 93 QVQLQESGGGMVQPGGSLRL sequence no. 2 SCAASGFDFG P, R, S 103 FW1 SEQ ID NO: 94 EVHLVESGGGLVRPGGSLRL sequence no. 3 SCAAFGFIFK P, R, S 103 FW1 SEQ ID NO: 95 QVQLAESGGGLVQPGGSLKL sequence no. 4 SCAASRTIVS P, R, S 103 FW1 SEQ ID NO: 96 QEHLVESGGGLVDIGGSLRL sequence no. 5 SCAASERIFS P, R, S 103 FW1 SEQ ID NO: 97 QVKLEESGGGLAQPGGSLRL sequence no. 6 SCVASGFTFS P, R, S 103 FW1 SEQ ID NO: 98 EVQLVESGGGLVQPGGSLRL sequence no. 7 SCVCVSSGCT P, R, S 103 FW1 SEQ ID NO: 99 EVQLVESGGGLALPGGSLTL sequence no. 8 SCVFSGSTFS and in which

-   iv) FR2 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-21 Representative FW2 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 FW2 SEQ ID NO: 102 WFRQAPGKEREFVA sequence no. 1 P, R, S 103 FW2 SEQ ID NO: 103 WVRQAPGKVLEWVS sequence no. 2 P, R, S 103 FW2 SEQ ID NO: 104 WVRRPPGKGLEWVS sequence no. 3 P, R, S 103 FW2 SEQ ID NO: 105 WIRQAPGKEREGVS sequence no. 4 P, R, S 103 FW2 SEQ ID NO: 106 WVRQYPGKEPEWVS sequence no. 5 P, R, S 103 FW2 SEQ ID NO: 107 WFRQPPGKEHEFVA sequence no. 6 P, R, S 103 FW2 SEQ ID NO: 108 WYRQAPGKRTELVA sequence no. 7 P, R, S 103 FW2 SEQ ID NO: 109 WLRQAPGQGLEWVS sequence no. 8 P, R, S 103 FW2 SEQ ID NO: 110 WLRQTPGKGLEWVG sequence no. 9 P, R, S 103 FW2 SEQ ID NO: 111 WVRQAPGKAEEFVS sequence no. 10 and in which:

-   v) FR3 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-22 Representative FW3 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 FW3 SEQ ID NO: 112 RFTISRDNAKNTVYLQMNS sequence no. 1 LKPEDTAVYYCAA P, R, S 103 FW3 SEQ ID NO: 113 RFTISRDNARNTLYLQMDS sequence no. 2 LIPEDTALYYCAR P, R, S 103 FW3 SEQ ID NO: 114 RFTISRDNAKNEMYLQMNN sequence no. 3 LKTEDTGVYVVCGA P, R, S 103 FW3 SEQ ID NO: 115 RFTISSDSNRNMIYLQMNN sequence no. 4 LKPEDTAVYYCAA P, R, S 103 FW3 SEQ ID NO: 116 RFTISRDNAKNMLYLHLNN sequence no. 5 LKSEDTAVYYCRR P, R, S 103 FW3 SEQ ID NO: 117 RFTISRDNAKKTVYLRLNS sequence no. 6 LNPEDTAVYSCNL P, R, S 103 FW3 SEQ ID NO: 118 RFKISRDNAKKTLYLQMNS sequence no. 7 LGPEDTAMYYCQR P, R, S 103 FW3 SEQ ID NO: 119 RFTVSRDNGKNTAYLRMNS sequence no. 8 LKPEDTADYYCAV and in which:

-   vi) FR4 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-23 Representative FW4 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 FW4 SEQ ID NO: 120 RGQGTQVTVSS sequence no. 1 P, R, S 103 FW4 SEQ ID NO: 121 LRGGTQVTVSS sequence no. 2 P, R, S 103 FW4 SEQ ID NO: 122 GNKGTLVTVSS sequence no. 3 P, R, S 103 FW4 SEQ ID NO: 123 SSPGTQVTVSS sequence no. 4 P, R, S 103 FW4 SEQ ID NO: 124 SSQGTLVTVSS sequence no. 5 P, R, S 103 FW4 SEQ ID NO: 125 RSRGIQVTVSS sequence no. 6 and in which:

-   vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably     as defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the P, R, S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

-   i) the amino acid residue at position 103 according to the Kabat     numbering is different from W;     and in which: -   ii) preferably the amino acid residue at position 103 according to     the Kabat numbering is P, R or S, and more preferably R;     and in which: -   iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the     Kabat numbering, has at least 80% amino acid identity with at least     one of the following amino acid sequences:

TABLE A-24 Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the P, R, S 103-group. P, R, S 103 FW1 SEQ ID NO: 100 VESGGGLVQAGGSLRLSC sequence no. 9 AASG P, R, S 103 FW1 SEQ ID NO: 101 AESGGGLVQPGGSLKLSC sequence no. 10 AASR and in which:

-   iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of     Nanobodies of the P, R, S 103 class;     and in which: -   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

In another preferred, but non-limiting aspect, the invention relates to a Nanobody as described above, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 2051-2325. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody and one or more of the sequences of SEQ ID NO's: 2051-2325, in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies can be as further described herein.

As already mentioned herein, another preferred but non-limiting aspect of the invention relates to a Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 2051-2325 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 2051-2325.

Also, in the above Nanobodies:

-   i) any amino acid substitution (when it is not a humanizing     substitution as defined herein) is preferably, and compared to the     corresponding amino acid sequence of SEQ ID NO's: 2051-2325, a     conservative amino acid substitution, (as defined herein);     and/or: -   ii) its amino acid sequence preferably contains either only amino     acid substitutions, or otherwise preferably no more than 5,     preferably no more than 3, and more preferably only 1 or 2 amino     acid deletions or insertions, compared to the corresponding amino     acid sequence of SEQ ID NO's: 2051-2325;     and/or -   iii) the CDR's may be CDR's that are derived by means of maturation,     for example starting from the CDR's of to the corresponding amino     acid sequence of SEQ ID NO's: 2051-2325.

Preferably, the CDR sequences and FR sequences in the Nanobodies of the invention are such that the Nanobodies of the invention (and polypeptides of the invention comprising the same):

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to         10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²         moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²         moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to         10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles         or more and more preferably 10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about         10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,         more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as         between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;         and/or such that they:     -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)         and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a         t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶         s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as         between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, CDR sequences and FR sequences present in the Nanobodies of the invention are such that the Nanobodies of the invention will bind to HER2 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

According to one non-limiting aspect of the invention, a Nanobody may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring human V_(H) domain, and in particular compared to the corresponding framework region of DP-47. More specifically, according to one non-limiting aspect of the invention, a Nanobody may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring human V_(H) domain, and in particular compared to the corresponding framework region of DP-47. Usually, a Nanobody will have at least one such amino acid difference with a naturally occurring V_(H) domain in at least one of FR2 and/or FR4, and in particular at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

Also, a humanized Nanobody of the invention may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring V_(HH) domain. More specifically, according to one non-limiting aspect of the invention, a humanized Nanobody may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring V_(HH) domain. Usually, a humanized Nanobody will have at least one such amino acid difference with a naturally occurring V_(HH) domain in at least one of FR2 and/or FR4, and in particular at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “analogs”) of the Nanobodies of the invention as defined herein, and in particular analogs of the Nanobodies of SEQ ID NO's 2051-2325. Thus, according to one aspect of the invention, the term “Nanobody of the invention” in its broadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the Nanobodies of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDR's. When such substitutions, insertions or deletions are made in one or more of the framework regions, they may be made at one or more of the Hallmark residues and/or at one or more of the other positions in the framework residues, although substitutions, insertions or deletions at the Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).

By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another V_(HH) domain (see Tables A-5 to A-8 for some non-limiting examples of such substitutions), although the invention is generally not limited thereto. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the Nanobody of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Nanobody of the invention (i.e. to the extent that the Nanobody is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express the Nanobody or polypeptide of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation (again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH) variability given in Tables A-5 to A-8 above, some amino acid residues in the framework regions are more conserved than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off) rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies of the invention.

The analogs are preferably also such that they retain the favourable properties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree of sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95% or 99% or more; and/or preferably have at most 20, preferably at most 10, even more preferably at most 5, such as 4, 3, 2 or only 1 amino acid difference (as defined herein), with one of the Nanobodies of SEQ ID NOs: 2051-2325.

Also, the framework sequences and CDR's of the analogs are preferably such that they are in accordance with the preferred aspects defined herein. More generally, as described herein, the analogs will have (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably an E at position 44, and more preferably E at position 44 and Rat position 45; and/or (c) P, R or S at position 103.

One preferred class of analogs of the Nanobodies of the invention comprise Nanobodies that have been humanized (i.e. compared to the sequence of a naturally occurring Nanobody of the invention). As mentioned in the background art cited herein, such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring V_(HH) with the amino acid residues that occur at the same position in a human V_(H) domain, such as a human V_(H)3 domain. Examples of possible humanizing substitutions or combinations of humanizing substitutions will be clear to the skilled person, for example from the Tables herein, from the possible humanizing substitutions mentioned in the background art cited herein, and/or from a comparison between the sequence of a Nanobody and the sequence of a naturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resulting humanized Nanobodies still retain the favourable properties of Nanobodies as defined herein, and more preferably such that they are as described for analogs in the preceding paragraphs. A skilled person will generally be able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible humanizing substitutions and determining their influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the invention may become more “human-like”, while still retaining the favorable properties of the Nanobodies of the invention as described herein. As a result, such humanized Nanobodies may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring V_(HH) domains. Again, based on the disclosure herein and optionally after a limited degree of routine experimentation, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring V_(HH) domains on the other hand.

The Nanobodies of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined herein) or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. One preferred humanizing substitution for Nanobodies of the “P,R,S-103 group” or the “KERE group” is Q108 into L108. Nanobodies of the “GLEW class” may also be humanized by a Q108 into L108 substitution, provided at least one of the other Hallmark residues contains a camelid (camelizing) substitution (as defined herein). For example, as mentioned above, one particularly preferred class of humanized Nanobodies has GLEW (SEQ ID NO: 2447) or a GLEW-like sequence at positions 44-47; P, R or S (and in particular R) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding the same, can be provided in any manner known per se. For example, the analogs can be obtained by providing a nucleic acid that encodes a naturally occurring V_(HH) domain, changing the codons for the one or more amino acid residues that are to be substituted into the codons for the corresponding desired amino acid residues (e.g. by site-directed mutagenesis or by PCR using suitable mismatch primers), expressing the nucleic acid/nucleotide sequence thus obtained in a suitable host or expression system; and optionally isolating and/or purifying the analog thus obtained to provide said analog in essentially isolated form (e.g. as further described herein). This can generally be performed using methods and techniques known per se, which will be clear to the skilled person, for example from the handbooks and references cited herein, the background art cited herein and/or from the further description herein. Alternatively, a nucleic acid encoding the desired analog can be synthesized in a manner known per se (for example using an automated apparatus for synthesizing nucleic acid sequences with a predefined amino acid sequence) and can then be expressed as described herein. Yet another technique may involve combining one or more naturally occurring and/or synthetic nucleic acid sequences each encoding a part of the desired analog, and then expressing the combined nucleic acid sequence as described herein. Also, the analogs can be provided using chemical synthesis of the pertinent amino acid sequence using techniques for peptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that the Nanobodies of the invention (including their analogs) can be designed and/or prepared starting from human V_(H) sequences (i.e. amino acid sequences or the corresponding nucleotide sequences), such as for example from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e. by introducing one or more camelizing substitutions (i.e. changing one or more amino acid residues in the amino acid sequence of said human V_(H) domain into the amino acid residues that occur at the corresponding position in a V_(HH) domain), so as to provide the sequence of a Nanobody of the invention and/or so as to confer the favourable properties of a Nanobody to the sequence thus obtained. Again, this can generally be performed using the various methods and techniques referred to in the previous paragraph, using an amino acid sequence and/or nucleotide sequence for a human V_(H) domain as a starting point.

Some preferred, but non-limiting camelizing substitutions can be derived from Tables A-5-A-8. It will also be clear that camelizing substitutions at one or more of the Hallmark residues will generally have a greater influence on the desired properties than substitutions at one or more of the other amino acid positions, although both and any suitable combination thereof are included within the scope of the invention. For example, it is possible to introduce one or more camelizing substitutions that already confer at least some the desired properties, and then to introduce further camelizing substitutions that either further improve said properties and/or confer additional favourable properties. Again, the skilled person will generally be able to determine and select suitable camelizing substitutions or suitable combinations of camelizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible camelizing substitutions and determining whether the favourable properties of Nanobodies are obtained or improved (i.e. compared to the original V_(H) domain). Generally, however, such camelizing substitutions are preferably such that the resulting an amino acid sequence at least contains (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably also an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103; and optionally one or more further camelizing substitutions. More preferably, the camelizing substitutions are such that they result in a Nanobody of the invention and/or in an analog thereof (as defined herein), such as in a humanized analog and/or preferably in an analog that is as defined in the preceding paragraphs.

As will also be clear from the disclosure herein, it is also within the scope of the invention to use parts or fragments, or combinations of two or more parts or fragments, of the Nanobodies of the invention as defined herein, and in particular parts or fragments of the Nanobodies of SEQ ID NO's: 2051-2325. Thus, according to one aspect of the invention, the term “Nanobody of the invention” in its broadest sense also covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention (including analogs thereof) have amino acid sequences in which, compared to the amino acid sequence of the corresponding full length Nanobody of the invention (or analog thereof), one or more of the amino acid residues at the N-terminal end, one or more amino acid residues at the C-terminal end, one or more contiguous internal amino acid residues, or any combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies of the invention.

Any part or fragment is preferably such that it comprises at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, more preferably at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues, of the amino acid sequence of the corresponding full length Nanobody of the invention.

Also, any part or fragment is such preferably that it comprises at least one of CDR1, CDR2 and/or CDR3 or at least part thereof (and in particular at least CDR3 or at least part thereof). More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least one other CDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connected by suitable framework sequence(s) or at least part thereof. More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least part of the two remaining CDR's, again preferably connected by suitable framework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect, such a part or fragment comprises at least CDR3, such as FR3, CDR3 and FR4 of the corresponding full length Nanobody of the invention, i.e. as for example described in the International application WO 03/050531 (Lasters et al.).

As already mentioned above, it is also possible to combine two or more of such parts or fragments (i.e. from the same or different Nanobodies of the invention), i.e. to provide an analog (as defined herein) and/or to provide further parts or fragments (as defined herein) of a Nanobody of the invention. It is for example also possible to combine one or more parts or fragments of a Nanobody of the invention with one or more parts or fragments of a human V_(H) domain.

According to one preferred aspect, the parts or fragments have a degree of sequence identity of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, such as at least 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs: 2051-2325.

The parts and fragments, and nucleic acid sequences encoding the same, can be provided and optionally combined in any manner known per se. For example, such parts or fragments can be obtained by inserting a stop codon in a nucleic acid that encodes a full-sized Nanobody of the invention, and then expressing the nucleic acid thus obtained in a manner known per se (e.g. as described herein). Alternatively, nucleic acids encoding such parts or fragments can be obtained by suitably restricting a nucleic acid that encodes a full-sized Nanobody of the invention or by synthesizing such a nucleic acid in a manner known per se. Parts or fragments may also be provided using techniques for peptide synthesis known per se.

The invention in its broadest sense also comprises derivatives of the Nanobodies of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g. enzymatical) modification, of the Nanobodies of the invention and/or of one or more of the amino acid residues that form the Nanobodies of the invention.

Examples of such modifications, as well as examples of amino acid residues within the Nanobody sequence that can be modified in such a manner (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. by covalent linking or in an other suitable manner) of one or more functional groups, residues or moieties into or onto the Nanobody of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the Nanobody of the invention. Example of such functional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. by covalent binding or in any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or the absorption of the Nanobody of the invention, that reduce the immunogenicity and/or the toxicity of the Nanobody of the invention, that eliminate or attenuate any undesirable side effects of the Nanobody of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties of the Nanobodies and/or polypeptides of the invention; or any combination of two or more of the foregoing. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv's and single domain antibodies), for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functional groups may for example be linked directly (for example covalently) to a Nanobody of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv's); reference is made to for example Chapman, Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov. 2, (2003) and in WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA.

Preferably, site-directed pegylation is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a Nanobody of the invention, a Nanobody of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of a Nanobody of the invention, all using techniques of protein engineering known per se to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example in the range of 20,000-80,000.

Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled Nanobody. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as ¹⁵²Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metal chelates or metallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I, ¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, ETA and other “sandwich assays”, etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair, Such a functional group may be used to link the Nanobody of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a Nanobody of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated Nanobody may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the Nanobody of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposomal formulations described by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the Nanobody of the invention.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation such a cell, the Nanobodies of the invention may also be linked to a toxin or to a toxic residue or moiety. Examples of toxic moieties, compounds or residues which can be linked to a Nanobody of the invention to provide—for example—a cytotoxic compound will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g. to study function-activity relationships). Reference is for example made to Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to HER2 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies of the invention.

As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of or comprise at least one Nanobody of the invention. By “essentially consist of” is meant that the amino acid sequence of the polypeptide of the invention either is exactly the same as the amino acid sequence of a Nanobody of the invention or corresponds to the amino acid sequence of a Nanobody of the invention which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwise influence the (biological) properties of the Nanobody and may or may not add further functionality to the Nanobody. For example, such amino acid residues:

-   -   can comprise an N-terminal Met residue, for example as result of         expression in a heterologous host cell or host organism.     -   may form a signal sequence or leader sequence that directs         secretion of the Nanobody from a host cell upon synthesis.         Suitable secretory leader peptides will be clear to the skilled         person, and may be as further described herein. Usually, such a         leader sequence will be linked to the N-terminus of the         Nanobody, although the invention in its broadest sense is not         limited thereto;     -   may form a sequence or signal that allows the Nanobody to be         directed towards and/or to penetrate or enter into specific         organs, tissues, cells, or parts or compartments of cells,         and/or that allows the Nanobody to penetrate or cross a         biological barrier such as a cell membrane, a cell layer such as         a layer of epithelial cells, a tumor including solid tumors, or         the blood-brain-barrier. Examples of such amino acid sequences         will be clear to the skilled person. Some non-limiting examples         are the small peptide vectors (“Pep-trans vectors”) described in         WO 03/026700 and in Temsamani et al., Expert Opin. Biol. Ther.,         1, 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9,         1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131         (2001), and the membrane translocator sequence described by Zhao         et al., Apoptosis, 8, 631-637 (2003). C-terminal and N-terminal         amino acid sequences for intracellular targeting of antibody         fragments are for example described by Cardinale et al.,         Methods, 34, 171 (2004). Other suitable techniques for         intracellular targeting involve the expression and/or use of         so-called “intrabodies” comprising a Nanobody of the invention,         as mentioned below;     -   may form a “tag”, for example an amino acid sequence or residue         that allows or facilitates the purification of the Nanobody, for         example using affinity techniques directed against said sequence         or residue. Thereafter, said sequence or residue may be removed         (e.g. by chemical or enzymatical cleavage) to provide the         Nanobody sequence (for this purpose, the tag may optionally be         linked to the Nanobody sequence via a cleavable linker sequence         or contain a cleavable motif). Some preferred, but non-limiting         examples of such residues are multiple histidine residues,         glutatione residues and a myc-tag (see for example SEQ ID NO:31         of WO 06/12282).     -   may be one or more amino acid residues that have been         functionalized and/or that can serve as a site for attachment of         functional groups. Suitable amino acid residues and functional         groups will be clear to the skilled person and include, but are         not limited to, the amino acid residues and functional groups         mentioned herein for the derivatives of the Nanobodies of the         invention.

According to another aspect, a polypeptide of the invention comprises a Nanobody of the invention, which is fused at its amino terminal end, at its carboxy terminal end, or both at its amino terminal end and at its carboxy terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein comprising said Nanobody of the invention and the one or more further amino acid sequences. Such a fusion will also be referred to herein as a “Nanobody fusion”.

The one or more further amino acid sequence may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the Nanobody, and may or may not add further functionality to the Nanobody or the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the Nanobody or the polypeptide of the invention.

For example, the further amino acid sequence may also provide a second binding site, which binding site may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including but not limited to the same protein, polypeptide, antigen, antigenic determinant or epitope against which the Nanobody of the invention is directed, or a different protein, polypeptide, antigen, antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv's and single domain antibodies). Reference is for example made to the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptides of the invention, compared to the Nanobody of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).

In particular, it has been described in the art that linking fragments of immunoglobulins (such as V_(H) domains) to serum albumin or to fragments thereof can be used to increase the half-life. Reference is for made to WO 00/27435 and WO 01/077137). According to the invention, the Nanobody of the invention is preferably either directly linked to serum albumin (or to a suitable fragment thereof) or via a suitable linker, and in particular via a suitable peptide linked so that the polypeptide of the invention can be expressed as a genetic fusion (protein). According to one specific aspect, the Nanobody of the invention may be linked, to a fragment of serum albumin that at least comprises the domain III of serum albumin or part thereof. Reference is for example made to WO 07/112,940 of Ablynx N.V.

Alternatively, the further amino acid sequence may provide a second binding site or binding unit that is directed against a serum protein (such as, for example, human serum albumin or another serum protein such as IgG), so as to provide increased half-life in serum. Such amino acid sequences for example include the Nanobodies described below, as well as the small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO 04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, as well as to EP 0 368 684, as well as to WO 08/028,977, WO 08/043,821, WO 08/043,822 by Ablynx N.V. mentioned herein and WO 08/068,280 by Ablynx N.V.

Such amino acid sequences may in particular be directed against serum albumin (and more in particular human serum albumin) and/or against IgG (and more in particular human IgG). For example, such amino acid sequences may be amino acid sequences that are directed against (human) serum albumin and amino acid sequences that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787) and/or amino acid sequences that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see again for example WO 06/0122787); amino acid sequences that have or can provide an increased half-life (see for example WO 08/028,977); amino acid sequences against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), reference is again made to WO 2008/028977); amino acid sequences that can bind to serum albumin in a pH independent manner (see for example WO2008/043821) and/or amino acid sequences that are conditional binders (see for example WO2008/043822).

According to another aspect, the one or more further amino acid sequences may comprise one or more parts, fragments or domains of conventional 4-chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, although usually less preferred, a Nanobody of the invention may be linked to a conventional (preferably human) V_(H) or V_(L) domain or to a natural or synthetic analog of a V_(H) or V_(L) domain, again optionally via a linker sequence (including but not limited to other (single) domain antibodies, such as the dAb's described by Ward et al.).

The at least one Nanobody may also be linked to one or more (preferably human) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linker sequence. For instance, a Nanobody linked to a suitable C_(H)1 domain could for example be used—together with suitable light chains—to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab′)₂ fragments, but in which one or (in case of an F(ab′)₂ fragment) one or both of the conventional V_(H) domains have been replaced by a Nanobody of the invention. Also, two Nanobodies could be linked to a C_(H)3 domain (optionally via a linker) to provide a construct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, one or more Nanobodies of the invention may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more C_(H)2 and/or C_(H)3 domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and/or may form (part of) and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 or IgG4), from IgE or from another human Ig such as IgA, IgD or IgM. For example, WO 94/04678 describes heavy chain antibodies comprising a Camelid V_(HH) domain or a humanized derivative thereof (i.e. a Nanobody), in which the Camelidae C_(H)2 and/or C_(H)3 domain have been replaced by human C_(H)2 and C_(H)3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a Nanobody and human C_(H)2 and C_(H)3 domains (but no C_(H)1 domain), which immunoglobulin has the effector function provided by the C_(H)2 and C_(H)3 domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the Nanobodies of the invention so as to provide an effector function will be clear to the skilled person, and may be chosen on the basis of the desired effector function(s). Reference is for example made to WO 04/058820, WO 99/42077, WO 02/056910 and WO 05/017148, as well as the review by Holliger and Hudson, supra and to the U.S. provisional application 61/005,324 entitled “Constructs comprising single variable domains and an Fc portion derived from IgE” filed on Dec. 4, 2007 (see also the International patent application by Ablynx N.V. entitled “Constructs comprising single variable domains and an Fc portion derived from IgE” with the same filing date as the present application) which is incorporated herein by reference.

The amino acids sequences or Nanobodies of the invention may, for example, be linked to an Fc portion that is capable of effecting one or more IgE-mediated immune responses and/or that is capable of binding to either the FcεRI receptor and/or the FcεRII receptor. The amino acid sequences or Nanobodies of the invention may be linked, optionally via a suitable linker or hinge region, to one or more constant domains, in which the constant domains from the first polypeptide chain and the second polypeptide chains together form an Fc portion that is capable of effecting one or more IgE-mediated immune responses and/or that is capable of binding to either the FcεRI receptor and/or the FcεRII receptor. In a preferred aspect, the Fc portion is capable of binding to FcεRI with an affinity (expressed as the K_(a) value) better than 10⁶ M⁻¹, preferably better than 10⁸ M⁻¹, more preferably better than 10⁹ M⁻¹, such as with a K_(a) value of about 10¹⁰ M⁻¹ or 10¹¹ M⁻¹. More preferably the Fc portion is capable of binding to FcεRI even with an affinity (expressed as the K_(a) value) better than 10⁶ M⁻¹, preferably better than 10⁷ M⁻¹, such as with a K_(a) value of about 10⁸ M⁻¹. Such an Fc portion may comprises one or more parts, fragments, amino acid stretches or domains of the Fc portion of IgE, preferably one or more of those parts, fragments, amino acid stretches or domains of the Fc portion of IgE that allow IgE to bind to its receptors. Preferably the Fc portion at least comprises C_(ε)4 (or a suitable part of fragment thereof), and optionally also comprises C_(ε)3 (or a suitable part of fragment thereof) and/or C_(ε)2 (or a suitable part of fragment thereof). Even more preferably, the Fc portion essentially consist of three constant domains, preferably all or essentially all derived from human Fc portions.

Coupling of a Nanobody of the invention to an Fc portion may also lead to an increased half-life, compared to the corresponding Nanobody of the invention. For some applications, the use of an Fc portion and/or of constant domains (i.e. C_(H)2 and/or C_(H)3 domains) that confer increased half-life without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more Nanobodies and one or more constant domains with increased half-life in vivo will be clear to the skilled person, and may for example comprise two Nanobodies linked to a C_(H)3 domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weight of more than 50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form a polypeptide of the invention, one or more amino acid sequences of the invention may be linked (optionally via a suitable linker or hinge region) to naturally occurring, synthetic or semisynthetic constant domains (or analogs, variants, mutants, parts or fragments thereof) that have a reduced (or essentially no) tendency to self-associate into dimers (i.e. compared to constant domains that naturally occur in conventional 4-chain antibodies). Such monomeric (i.e. not self-associating) Fc chain variants, or fragments thereof, will be clear to the skilled person. For example, Helm et al., J Biol Chem 1996 271 7494, describe monomeric Fcε chain variants that can be used in the polypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they are still capable of binding to the complement or the relevant Fc receptor(s) (depending on the Fc portion from which they are derived), and/or such that they still have some or all of the effector functions of the Fc portion from which they are derived (or at a reduced level still suitable for the intended use). Alternatively, in such a polypeptide chain of the invention, the monomeric Fc chain may be used to confer increased half-life upon the polypeptide chain, in which case the monomeric Fc chain may also have no or essentially no effector functions.

Bivalent/multivalent, bispecific/multispecific or biparatopic/multiparatopic polypeptides of the invention may also be linked to Fc portions, in order to provide polypeptide constructs of the type that is described in U.S. provisional application 61/005,331 entitled “Immunoglobulin constructs” filed on Dec. 4, 2007 (see also the International patent application by Ablynx N.V. entitled “Immunoglobulin constructs” with the same filing date as the present application) which is incorporated herein by reference.

The invention, for example also relates to compounds or constructs that comprises an Fc portion that is linked, optionally via a suitable linker or hinge region, to a pair of first amino acid sequences of the invention (preferably Nanobodies of the invention), which are linked, optionally via a suitable linker, to a pair of second amino acid sequences of the invention (preferably Nanobodies of the invention), wherein:

-   -   both of the first amino acid sequences of the invention         (preferably Nanobodies of the invention) are directed against a         first epitope, antigenic determinant, part, domain or subunit on         HER2; and     -   both of the amino acid sequences of the invention (preferably         Nanobodies of the invention) are directed against a second         epitope, antigenic determinant, part, domain or subunit on HER2         which is the same or different from said first epitope,         antigenic determinant, part, domain or subunit.         Such compounds or constructs of the invention may, for example         be directed against the Herceptin® binding site on HER-2 (and in         particular against domain IV of HER2, and more in particular         against the C-terminus of domain IV of HER2) and/or are         compounds or constructs that are capable of competing with         Herceptin® for binding to HER-2; they may be directed against         the Omnitarg® binding site on HER-2 (and in particular against         domain II of HER2, and more in particular against the middle of         domain II of HER2) and/or are compounds or constructs that are         capable of competing with Omnitarg® for binding to HER-2; or         they may be directed against the Herceptin® binding site on         HER-2 (and in particular against domain IV of HER2, and more in         particular against the C-terminus of domain IV of HER2) and/or         are compounds or constructs that are capable of competing with         Herceptin® for binding to HER-2 and simultaneously be directed         against the Omnitarg® binding site on HER-2 (and in particular         against domain II of HER2, and more in particular against the         middle of domain II of HER2) and/or are compounds or constructs         that are capable of competing with Omnitarg® for binding to         HER-2.

The further amino acid sequences may also form a signal sequence or leader sequence that directs secretion of the Nanobody or the polypeptide of the invention from a host cell upon synthesis (for example to provide a pre-, pro- or prepro-form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention).

The further amino acid sequence may also form a sequence or signal that allows the Nanobody or polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody or polypeptide of the invention to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Suitable examples of such amino acid sequences will be clear to the skilled person, and for example include, but are not limited to, the “Peptrans” vectors mentioned above, the sequences described by Cardinale et al. and the amino acid sequences and antibody fragments known per se that can be used to express or produce the Nanobodies and polypeptides of the invention as so-called “intrabodies”, for example as described in WO 94/02610, WO 95/22618, U.S. Pat. No. 7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag; and in Kontermann, Methods 34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation of such a cell, the Nanobodies of the invention may also be linked to a (cyto)toxic protein or polypeptide. Examples of such toxic proteins and polypeptides which can be linked to a Nanobody of the invention to provide—for example—a cytotoxic polypeptide of the invention will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or more further amino acid sequences comprise at least one further Nanobody, so as to provide a polypeptide of the invention that comprises at least two, such as three, four, five or more Nanobodies, in which said Nanobodies may optionally be linked via one or more linker sequences (as defined herein). Polypeptides of the invention that comprise two or more Nanobodies, of which at least one is a Nanobody of the invention, will also be referred to herein as “multivalent” polypeptides of the invention, and the Nanobodies present in such polypeptides will also be referred to herein as being in a “multivalent format”. For example a “bivalent” polypeptide of the invention comprises two Nanobodies, optionally linked via a linker sequence, whereas a “trivalent” polypeptide of the invention comprises three Nanobodies, optionally linked via two linker sequences; etc.; in which at least one of the Nanobodies present in the polypeptide, and up to all of the Nanobodies present in the polypeptide, is/are a Nanobody of the invention.

In a multivalent polypeptide of the invention, the two or more Nanobodies may be the same or different, and may be directed against the same antigen or antigenic determinant (for example against the same part(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. For example, a bivalent polypeptide of the invention may comprise (a) two identical Nanobodies; (b) a first Nanobody directed against a first antigenic determinant of a protein or antigen and a second Nanobody directed against the same antigenic determinant of said protein or antigen which is different from the first Nanobody; (c) a first Nanobody directed against a first antigenic determinant of a protein or antigen and a second Nanobody directed against another antigenic determinant of said protein or antigen; or (d) a first Nanobody directed against a first protein or antigen and a second Nanobody directed against a second protein or antigen (i.e. different from said first antigen). Similarly, a trivalent polypeptide of the invention may, for example and without being limited thereto. comprise (a) three identical Nanobodies; (b) two identical Nanobody against a first antigenic determinant of an antigen and a third Nanobody directed against a different antigenic determinant of the same antigen; (c) two identical Nanobody against a first antigenic determinant of an antigen and a third Nanobody directed against a second antigen different from said first antigen; (d) a first Nanobody directed against a first antigenic determinant of an antigen, a second Nanobody directed against a second antigenic determinant of said antigen and a third Nanobody directed against a third antigenic determinant of the same antigen; (e) a first Nanobody directed against a first antigenic determinant of a first antigen, a second Nanobody directed against a second antigenic determinant of said first antigen and a third Nanobody directed against a second antigen different from said first antigen; or (f) a first Nanobody directed against a first antigen, a second Nanobody directed against a second antigen different from said first antigen, and a third Nanobody directed against a third antigen different from said first and second antigen.

Polypeptides of the invention that contain at least two Nanobodies, in which at least one Nanobody is directed against a first antigenic determinant on HER2 and at least one Nanobody is directed against a second antigenic determinant on HER2 will also be referred to as “multiparatopic” polypeptides of the invention, and the Nanobodies present in such polypeptides will also be referred to herein as being in a “multiparatopic format”. Thus, for example, a “biparatopic” polypeptide of the invention is a polypeptide that comprises at least one Nanobody directed against a first antigenic determinant on HER2 and at least one further Nanobody directed against a second antigenic determinant on HER2, whereas a “triparatopic” polypeptide of the invention is a polypeptide that comprises at least one Nanobody directed against a first antigenic determinant on HER2, at least one further Nanobody directed against a second antigenic determinant on HER2 and at least one further Nanobody directed against a third antigenic determinant on HER2; etc.

Accordingly, in its simplest form, a biparatopic polypeptide of the invention is a bivalent polypeptide of the invention (as defined herein), comprising a first Nanobody directed against a first antigenic determinant on HER2, and a second Nanobody directed against a second antigenic determinant on HER2, in which said first and second Nanobody may optionally be linked via a linker sequence (as defined herein); whereas a triparatopic polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined herein), comprising a first Nanobody directed against a first antigenic determinant on HER2, a second. Nanobody directed against a second antigenic determinant on HER2 and a third Nanobody directed against a third antigenic determinant on HER2, in which said first, second and third Nanobody may optionally be linked via one or more, and in particular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, the invention is not limited thereto, in the sense that a multiparatopic polypeptide of the invention may comprise at least one Nanobody against a first antigenic determinant on HER2, and any number of Nanobodies directed against one or more other antigenic determinants on HER2.

Polypeptides of the invention that contain at least two Nanobodies, in which at least one Nanobody is directed against a first antigen (i.e. against HER2) and at least one Nanobody is directed against a second antigen (i.e. different from HER2), will also be referred to as “multispecific” polypeptides of the invention, and the Nanobodies present in such polypeptides will also be referred to herein as being in a “multispecific format”. Thus, for example, a “bispecific” polypeptide of the invention is a polypeptide that comprises at least one Nanobody directed against a first antigen (i.e. HER2) and at least one further Nanobody directed against a second antigen (i.e. different from HER2), whereas a “trispecific” polypeptide of the invention is a polypeptide that comprises at least one Nanobody directed against a first antigen (i.e. HER2), at least one further Nanobody directed against a second antigen (i.e. different from HER2) and at least one further Nanobody directed against a third antigen (i.e. different from both HER2, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of the invention is a bivalent polypeptide of the invention (as defined herein), comprising a first Nanobody directed against HER2, and a second Nanobody directed against a second antigen, in which said first and second Nanobody may optionally be linked via a linker sequence (as defined herein); whereas a trispecific polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined herein), comprising a first Nanobody directed against HER2, a second Nanobody directed against a second antigen and a third Nanobody directed against a third antigen, in which said first, second and third Nanobody may optionally be linked via one or more, and in particular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, the invention is not limited thereto, in the sense that a multispecific polypeptide of the invention may comprise at least one Nanobody against HER2, and any number of Nanobodies directed against one or more antigens different from HER2.

Furthermore, although it is encompassed within the scope of the invention that the specific order or arrangement of the various Nanobodies in the polypeptides of the invention may have some influence on the properties of the final polypeptide of the invention (including but not limited to the affinity, specificity or avidity for HER2, or against the one or more other antigens), said order or arrangement is usually not critical and may be suitably chosen by the skilled person, optionally after some limited routine experiments based on the disclosure herein. Thus, when reference is made to a specific multivalent or multispecific polypeptide of the invention, it should be noted that this encompasses any order or arrangements of the relevant Nanobodies, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that the polypeptides of the invention contain two or more Nanobodies and one or more further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or more V_(HH) domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to for example WO 96/34103 and WO 99/23221. Some other examples of some specific multispecific and/or multivalent polypeptide of the invention can be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody of the invention and at least one Nanobody that provides for an increased half-life. Such Nanobodies may for example be Nanobodies that are directed against a serum protein, and in particular a human serum protein, such as human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one of the serum proteins listed in WO 04/003019. Of these, Nanobodies that can bind to serum albumin (and in particular human serum albumin) or to IgG (and in particular human IgG, see for example Nanobody VH-1 described in the review by Muyldermans, supra) are particularly preferred (although for example, for experiments in mice or primates, Nanobodies against or cross-reactive with mouse serum albumin (MSA) or serum albumin from said primate, respectively, can be used. However, for pharmaceutical use, Nanobodies against human serum albumin or human IgG will usually be preferred). Nanobodies that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies directed against serum albumin that are described in WO 04/041865, in WO 06/122787 and in the further patent applications by Ablynx N.V., such as those mentioned above.

For example, the some preferred Nanobodies that provide for increased half-life for use in the present invention include Nanobodies that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787); Nanobodies that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see for example WO 06/0122787); Nanobodies that have or can provide an increased half-life (see for example WO 08/028,977 by Ablynx N.V.); Nanobodies against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) (see for example WO 08/028,977 by Ablynx N.V.); Nanobodies that can bind to serum albumin in a pH independent manner (see for example WO 08/043,821 by Ablynx N.V.) and/or Nanobodies that are conditional binders (see for example WO 08/043,822 by Ablynx N.V.).

Some particularly preferred Nanobodies that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) is particularly preferred.

Some preferred, but non-limiting examples of polypeptides of the invention that comprise at least one Nanobody of the invention and at least one Nanobody that provides for increased half-life are given in SEQ ID NOs: 2331-2335.

According to a specific, but non-limiting aspect of the invention, the polypeptides of the invention contain, besides the one or more Nanobodies of the invention, at least one Nanobody against human serum albumin.

Generally, any polypeptides of the invention with increased half-life that contain one or more Nanobodies of the invention, and any derivatives of Nanobodies of the invention or of such polypeptides that have an increased half-life, preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding Nanobody of the invention per se. For example, such a derivative or polypeptides with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding Nanobody of the invention per se.

In a preferred, but non-limiting aspect of the invention, such derivatives or polypeptides may exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, such derivatives or polypeptides may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable of binding to one or more molecules which can increase the half-life of the polypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and their half-life increased by binding to molecules which resist degradation and/or clearance or sequestration. Typically, such molecules are naturally occurring proteins which themselves have a long half-life in vivo.

Another preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody of the invention and at least one Nanobody that directs the polypeptide of the invention towards, and/or that allows the polypeptide of the invention to penetrate or to enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such Nanobodies include Nanobodies that are directed towards specific cell-surface proteins, markers or epitopes of the desired organ, tissue or cell (for example cell-surface markers associated with tumor cells), and the single-domain brain targeting antibody fragments described in WO 02/057445 and WO 06/040153, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies and the one or more polypeptides may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent, multiparatopic and multispecific polypeptides will be clear to the skilled person, and may generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing proteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers that are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, its should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent V_(H) and V_(L) domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each Nanobody by itself forms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 75, preferably between 1 and 60, more preferably between 1 and 50, even more preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (gly_(x)ser_(y))_(z), such as (for example (gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30, GS15, GS9 and GS7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/040153 and WO 06/122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such as AAA), as well as the linkers GS35, GS30 (SEQ ID NO: 85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, polyethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for HER2, or for one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

For example, in multivalent polypeptides of the invention that comprise Nanobodies directed against a multimeric antigen (such as a multimeric receptor or other protein), the length and flexibility of the linker are preferably such that it allows each Nanobody of the invention present in the polypeptide to bind to the antigenic determinant on each of the subunits of the multimer.

Similarly, in a multiparatopic polypeptide of the invention that comprises Nanobodies directed against two or more different antigenic determinants on the same antigen (for example against different epitopes of an antigen and/or against different subunits of a multimeric receptor or protein), the length and flexibility of the linker are preferably such that, when the multiparatopic polypeptide binds to HER-2, at least two and preferably all of the Nanobodies that are present in the multiparatopic polypeptide can (simultaneously) bind to each of their intended antigenic determinants, epitopes, parts or domains, most preferably so as to allow binding with increased avidity and also intramolecular binding and/or recognition. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

For example, as further described herein, some of the most preferred multiparatopic polypeptides of the invention comprise (i) at least one amino acid sequence of the invention (and in particular at least one Nanobody) that is directed against the Omnitarg binding site on HER2 (and in particular against domain II of HER2, and more in particular against the middle of domain II of HER2) and/or that is capable of competing with Omnitarg for binding to HER-2; and at least one amino acid sequence of the invention (and in particular at least one Nanobody) that is directed against the Herceptin® binding site on HER2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2) and/or that is capable of competing with Herceptin® for binding to HER-2. In such a preferred multiparatopic polypeptide of the invention, the linker is most preferably such that the multiparatopic polypeptide of the invention is capable of (simultaneously) binding to both the Omnitarg binding site on HER2 (and in particular against domain II of HER2, and more in particular against the middle of domain of HER2) as well as the Herceptin® binding site on HER2 (and in particular against domain IV of HER2, and more in particular against the C-terminus of domain IV of HER2), again most preferably so as to allow binding with increased avidity and also intramolecular binding and/or recognition. Such multiparatopic polypeptides of the invention with such a linker form a particularly preferred aspect of the invention, and examples of such a linker are given in the Examples below. For example, when such a linker is a Gly-Ser linker (for example, a Gly-Ser linker as described in the Examples), it preferably has a length of at least 15 amino acid residues, such as at least 20 or at least 30 amino acid residues. The maximum length is not especially critical, but for practical considerations (such as ease of cloning and expression) the linker is preferably no longer than 75 amino acid residues, more preferably less than 50 amino acid residues. For example, Gly-Ser linkers (such as the Gly-Ser linkers as described in the Examples) of between 20 and 40 amino acid residues, such as about 25, 30 or 35 amino acid residues, may be particularly suited. Based on the disclosure herein, the skilled person will be able to determine other suitable linkers, it being understood that the optimal length of each linker may also depend on the amino acid composition of the linker that is envisaged for use.

Optimal linker lengths in biparatopic, triparatopic or multiparatopic polypeptides of the invention can, for example, be designed in silico with any method for protein design known in the art or disclosed herein (see, e.g. the Example section). Optimal linker lengths, far example obtained by in silico design, can further be verified experimentally by binding and competition assays as will be known to the skilled person and/or described herein (see e.g. the example section). Optimal linker lengths in biparatopic, triparatopic or multiparatopic polypeptides may also be determined using the screening method for determining optimal linker length as described herein.

The choice of linker length in biparatopic, triparatopic or multiparatopic polypeptides of the invention can also be such that only a limited epitope space on the antigen is covered. Linker length restriction can, for example, help to avoid targeting epitopes which should not be neutralized (e.g. those essential for a function of the antigen) or to target regions relatively adjacent to a first ‘guiding’ Nanobody.

The choice of the format (N- or C-terminal position of the different Nanobodies) of the biparatopic, triparatopic or multiparatopic polypeptides of the invention and linker length can also be used to obtain molecules that bind avidly to the target antigen (via two, or more, binding sites), yet are purposely not agonistic. By optimising the format and linker length and composition, the binding sites can be positioned in such way that simultaneous binding of two or more Nanobodies to the same target antigen (i.e. intramolecular binding) will be highly favoured compared to binding to separate antigens in proximity of one another (intermolecular binding, such as e.g. on a cell surface). This could, for example, reduce the chance on agonism (which might not be desired in a good therapeutic compound). Screening and/or selection methods and assays are known to the skilled person and/or described herein that allow for the isolation of avidly binding domains positioned in relation to one another and to the antigen of interest in such way as to have an antagonistic function only.

In another aspect of the invention, biparatopic, triparatopic or multiparatopic polypeptides of the invention can also be selected to be purposely agonistic. For example, a combination of two identical or two different Nanobodies that bind to the Herceptin®-binding site and are genetically fused to one another can be agonistic (e.g. 2D3-2D3 or 2D3 fused to other Herceptin®-competing Nanobodies). The current invention also provides a way to select for such agonistic biparatopic, triparatopic or multiparatopic polypeptides of the invention using appropriate screening and/or selection procedures of members of multiparatopic libraries. Agonists could, for example, be desired and/or interesting for triggering certain receptors.

It is also within the scope of the invention that the linker(s) used confer one or more other favourable properties or functionality to the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g. as described herein for the derivatives of the Nanobodies of the invention). For example, linkers containing one or more charged amino acid residues (see Table A-2 above) can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of the invention will be a linear polypeptide. However, the invention in its broadest sense is not limited thereto. For example, when a polypeptide of the invention comprises three of more Nanobodies, it is possible to link them by use of a linker with three or more “arms”, which each “arm” being linked to a Nanobody, so as to provide a “star-shaped” construct. It is also possible, although usually less preferred, to use circular constructs.

The invention also comprises derivatives of the polypeptides of the invention, which may be essentially analogous to the derivatives of the Nanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentially consist” of a polypeptide of the invention (in which the wording “essentially consist of” has essentially the same meaning as indicated hereinabove).

According to one aspect of the invention, the polypeptide of the invention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from the further description herein. For example, the Nanobodies and polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for preparing the amino acid sequences, Nanobodies, polypeptides and nucleic acids include the methods and techniques described herein.

As will be clear to the skilled person, one particularly useful method for preparing an amino acid sequence, Nanobody and/or a polypeptide of the invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also     referred to herein as a “host of the invention”) or in another     suitable expression system of a nucleic acid that encodes said amino     acid sequence, Nanobody or polypeptide of the invention (also     referred to herein as a “nucleic acid of the invention”), optionally     followed by: -   ii) isolating and/or purifying the amino acid sequence, Nanobody or     polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under     conditions that are such that said host of the invention expresses     and/or produces at least one amino acid sequence, Nanobody and/or     polypeptide of the invention; optionally followed by: -   ii) isolating and/or purifying the amino acid sequence, Nanobody or     polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or double stranded DNA or RNA, and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism).

According to one aspect of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. To provide analogs, nucleotide sequences encoding naturally occurring V_(HH) domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analog. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least one nucleotide sequence encoding a Nanobody and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more “mismatched” primers, using for example a sequence of a naturally occurring form of HER2 as a template. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and are preferably double-stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).

In a preferred but non-limiting aspect, a genetic construct of the invention comprises

-   i) at least one nucleic acid of the invention; operably connected to -   ii) one or more regulatory elements, such as a promoter and     optionally a suitable terminator;     and optionally also -   iii) one or more further elements of genetic constructs known per     se;     in which the terms “regulatory element”, “promoter”, “terminator”     and “operably connected” have their usual meaning in the art (as     further described herein); and in which said “further elements”     present in the genetic constructs may for example be 3′- or 5′-UTR     sequences, leader sequences, selection markers, expression     markers/reporter genes, and/or elements that may facilitate or     increase (the efficiency of) transformation or integration. These     and other suitable elements for such genetic constructs will be     clear to the skilled person, and may for instance depend upon the     type of construct used, the intended host cell or host organism; the     manner in which the nucleotide sequences of the invention of     interest are to be expressed (e.g. via constitutive, transient or     inducible expression); and/or the transformation technique to be     used. For example, regulatory sequences, promoters and terminators     known per se for the expression and production of antibodies and     antibody fragments (including but not limited to (single) domain     antibodies and ScFv fragments) may be used in an essentially     analogous manner.

Preferably, in the genetic constructs of the invention, said at least one nucleic acid of the invention and said regulatory elements, and optionally said one or more further elements, are “operably linked” to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being “under the control of” said promotor). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.

Preferably, the regulatory and further elements of the genetic constructs of the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism.

For instance, a promoter, enhancer or terminator should be “operable” in the intended host cell or host organism, by which is meant that (for example) said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence—e.g. a coding sequence—to which it is operably linked (as defined herein).

Some particularly preferred promoters include, but are not limited to, promoters known per se for the expression in the host cells mentioned herein; and in particular promoters for the expression in the bacterial cells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriate selection conditions—host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or host organism—it allows for the desired post-translational modifications and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell. A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism. Leader sequences may not be required for expression in a bacterial cell. For example, leader sequences known per se for the expression and production of antibodies and antibody fragments (including but not limited to single domain antibodies and ScFv fragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the host cell or host organism—it allows for detection of the expression of (a gene or nucleotide sequence present on) the genetic construct. An expression marker may optionally also allow for the localisation of the expressed product, e.g. in a specific part or organelle of a cell and/or in (a) specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism. Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP.

Some preferred, but non-limiting examples of suitable promoters, terminator and further elements include those that can be used for the expression in the host cells mentioned herein; and in particular those that are suitable for expression in bacterial cells, such as those mentioned herein and/or those used in the Examples below. For some (further) non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention—such as terminators, transcriptional and/or translational enhancers and/or integration factors—reference is made to the general handbooks such as Sambrook et al. and Ausubel et al. mentioned above, as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320. WO 98/06737, WO 98/21355, U.S. Pat. No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person. Reference is also made to the general background art cited above and the further references cited herein.

The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the Examples below, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the amino acid sequence, Nanobody or polypeptide of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative         strains such as strains of Escherichia coli; of Proteus, for         example of Proteus mirabilis; of Pseudomonas, for example of         Pseudomonas fluorescens; and gram-positive strains such as         strains of Bacillus, for example of Bacillus subtilis or of         Bacillus brevis; of Streptomyces, for example of Streptomyces         lividans; of Staphylococcus, for example of Staphylococcus         carnosus; and of Lactococcus, for example of Lactococcus lactis;     -   a fungal cell, including but not limited to cells from species         of Trichoderma, for example from Trichoderma reesei; of         Neurospora, for example from Neurospora crassa; of Sordaria, for         example from Sordaria macrospora; of Aspergillus, for example         from Aspergillus niger or from Aspergillus sojae; or from other         filamentous fungi;     -   a yeast cell, including but not limited to cells from species of         Saccharomyces, for example of Saccharomyces cerevisiae; of         Schizosaccharomyces, for example of Schizosaccharomyces pombe;         of Pichia, for example of Pichia pastoris or of Pichia         methanolica; of Hansenula, for example of Hansenula polymorpha;         of Kluyveromyces, for example of Kluyveromyces lactis; of         Arxula, for example of Arxula adeninivorans; of Yarrowia, for         example of Yarrowia lipolytica;     -   an amphibian cell or cell line, such as Xenopus oocytes;     -   an insect-derived cell or cell line, such as cells/cell lines         derived from lepidoptera, including but not limited to         Spodoptera SF9 and Sf21 cells or cells/cell lines derived from         Drosophila, such as Schneider and Kc cells;     -   a plant or plant cell, for example in tobacco plants; and/or     -   a mammalian cell or cell line, for example a cell or cell line         derived from a human, a cell or a cell line from mammals         including but not limited to CHO-cells, BHK-cells (for example         BHK-21 cells) and human cells or cell lines such as HeLa, COS         (for example COS-7) and PER.C6 cells;         as well as all other hosts or host cells known per se for the         expression and production of antibodies and antibody fragments         (including but not limited to (single) domain antibodies and         ScFv fragments), which will be clear to the skilled person.         Reference is also made to the general background art cited         hereinabove, as well as to for example WO 94/29457; WO 96/34103;         WO 99/42077; Frenken et al., (1998), supra; Riechmann and         Muyldermans, (1999), supra; van der Linden, (2000), supra;         Thomassen et al., (2002), supra; Joosten et al., (2003), supra;         Joosten et al., (2005), supra; and the further references cited         herein.

The amino acid sequences, Nanobodies and polypeptides of the invention can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g. as a gene therapy). For this purpose, the nucleotide sequences of the invention may be introduced into the cells or tissues in any suitable way, for example as such (e.g. using liposomes) or after they have been inserted into a suitable gene therapy vector (for example derived from retroviruses such as adenovirus, or parvoviruses such as adeno-associated virus). As will also be clear to the skilled person, such gene therapy may be performed in vivo and/or in situ in the body of a patient by administering a nucleic acid of the invention or a suitable gene therapy vector encoding the same to the patient or to specific cells or a specific tissue or organ of the patient; or suitable cells (often taken from the body of the patient to be treated, such as explanted lymphocytes, bone marrow aspirates or tissue biopsies) may be treated in vitro with a nucleotide sequence of the invention and then be suitably (re-)introduced into the body of the patient. All this can be performed using gene therapy vectors, techniques and delivery systems which are well known to the skilled person, and for example described in Culver, K. W., “Gene Therapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.); Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.; 811 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,589,546; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. For example, in situ expression of ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has been described in the art.

For expression of the Nanobodies in a cell, they may also be expressed as so-called “intrabodies”, as for example described in WO 94/02610, WO 95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag; and in Kontermann, Methods 34, (2004), 163-170.

The amino acid sequences, Nanobodies and polypeptides of the invention can for example also be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep (see for example U.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No. 6,849,992 for general techniques for introducing transgenes into mammals), in plants or parts of plants including but not limited to their leaves, flowers, fruits, seed, roots or turbers (for example in tobacco, maize, soybean or alfalfa) or in for example pupae of the silkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear to the skilled person. Some preferred, but non-limiting examples include expression in the wheat germ system; in rabbit reticulocyte lysates; or in the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies is that the polypeptides based thereon can be prepared through expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, host cells, regulatory elements, etc., will be clear to the skilled person, for example from the references cited above. It should however be noted that the invention in its broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expression system, such as a bacterial expression system, is used that provides the polypeptides of the invention in a form that is suitable for pharmaceutical use, and such expression systems will again be clear to the skilled person. As also will be clear to the skilled person, polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the (industrial) production of Nanobodies or Nanobody-containing protein therapeutics include strains of E. coli, Pichia pastoris, S. cerevisiae that are suitable for large scale expression/production/fermentation, and in particular for large scale pharmaceutical (i.e. GMP grade) expression/production/fermentation. Suitable examples of such strains will be clear to the skilled person. Such strains and production/expression systems are also made available by companies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian, cell lines, in particular Chinese hamster ovary (CHO) cells, can be used for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also made available by some of the companies mentioned above.

The choice of the specific expression system would depend in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a Nanobody-containing recombinant protein for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e. the kind, number and position of residues attached) will depend on the cell or cell line that is used for the expression. Preferably, either a human cell or cell line is used (i.e. leading to a protein that essentially has a human glycosylation pattern) or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same as human glycosylation or at least mimics human glycosylation. Generally, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotes such as yeast usually leads to a glycosylation pattern that differs from human glycosylation. Nevertheless, it should be understood that all the foregoing host cells and expression systems can be used in the invention, depending on the desired amino acid sequence, Nanobody or polypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is glycosylated. According to another non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is non-glycosylated.

According to one preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale pharmaceutical production, such as cells of the strains mentioned above.

According to another preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a mammalian cell, in particular in a human cell or in a cell of a human cell line, and more in particular in a human cell or in a cell of a human cell line that is suitable for large scale pharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acid sequences, Nanobodies and the polypeptides of the invention, the amino acid sequences, Nanobodies and polypeptides of the invention can be produced either intracellularly (e.g. in the cytosol, in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. When eukaryotic host cells are used, extracellular production is usually preferred since this considerably facilitates the further isolation and downstream processing of the Nanobodies and proteins obtained. Bacterial cells such as the strains of E. coli mentioned above normally do not secrete proteins extracellularly, except for a few classes of proteins such as toxins and hemolysin, and secretory production in E. coli refers to the translocation of proteins across the inner membrane to the periplasmic space. Periplasmic production provides several advantages over cytosolic production. For example, the N-terminal amino acid sequence of the secreted product can be identical to the natural gene product after cleavage of the secretion signal sequence by a specific signal peptidase. Also, there appears to be much less protease activity in the periplasm than in the cytoplasm. In addition, protein purification is simpler due to fewer contaminating proteins in the periplasm. Another advantage is that correct disulfide bonds may form because the periplasm provides a more oxidative environment than the cytoplasm. Proteins overexpressed in E. coli are often found in insoluble aggregates, so-called inclusion bodies. These inclusion bodies may be located in the cytosol or in the periplasm; the recovery of biologically active proteins from these inclusion bodies requires a denaturation/refolding process. Many recombinant proteins, including therapeutic proteins, are recovered from inclusion bodies. Alternatively, as will be clear to the skilled person, recombinant strains of bacteria that have been genetically modified so as to secrete a desired protein, and in particular an amino acid sequence, Nanobody or a polypeptide of the invention, can be used.

Thus, according to one non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is an amino acid sequence, Nanobody or polypeptide that has been produced intracellularly and that has been isolated from the host cell, and in particular from a bacterial cell or from an inclusion body in a bacterial cell. According to another non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is an amino acid sequence, Nanobody or polypeptide that has been produced extracellularly, and that has been isolated from the medium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cells include,

-   -   for expression in E. coli: lac promoter (and derivatives thereof         such as the lacUV5 promoter); arabinose promoter; left-(PL) and         rightward (PR) promoter of phage lambda; promoter of the trp         operon; hybrid lac/trp promoters (tac and trc); T7-promoter         (more specifically that of T7-phage gene 10) and other T-phage         promoters; promoter of the Tn10 tetracycline resistance gene;         engineered variants of the above promoters that include one or         more copies of an extraneous regulatory operator sequence; for         expression in S. cerevisiae: constitutive: ADH1 (alcohol         dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),         GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1         (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:         GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol         dehydrogenase 2), PHO5 (acid phosphatase), CUP 1 (copper         metallothionein); heterologous: CaMV (cauliflower mosaic virus         ³⁵S promoter);     -   for expression in Pichia pastoris: the AOX1 promoter (alcohol         oxidase I);     -   for expression in mammalian cells: human cytomegalovirus (hCMV)         immediate early enhancer/promoter; human cytomegalovirus (hCMV)         immediate early promoter variant that contains two tetracycline         operator sequences such that the promoter can be regulated by         the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)         promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)         enhancer/promoter; elongation factor 1α (hEF-1α) promoter from         human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1         long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cells include:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),         pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSGS (Stratagene),         EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),         pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo         (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and         1ZD35 (ATCC 37565), as well as viral-based expression systems,         such as those based on adenovirus;     -   vectors for expression in bacterial cells: pET vectors (Novagen)         and pQE vectors (Qiagen);     -   vectors for expression in yeast or other fungal cells: pYES2         (Invitrogen) and Pichia expression vectors (Invitrogen);     -   vectors for expression in insect cells: pBlueBacII (Invitrogen)         and other baculovirus vectors     -   vectors for expression in plants or plant cells: for example         vectors based on cauliflower mosaic virus or tobacco mosaic         virus, suitable strains of Agrobacterium, or Ti-plasmid based         vectors.

Some preferred, but non-limiting secretory sequences for use with these host cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,         OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the         like; TAT signal peptide, hemolysin C-terminal secretion signal;     -   for use in yeast: α-mating factor prepro-sequence, phosphatase         (pho1), invertase (Suc), etc.;     -   for use in mammalian cells: indigenous signal in case the target         protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal         peptide; etc.

Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above.

After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the amino acid sequence of the invention, e.g. using specific antibodies.

The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g. under suitable conditions), an amino acid sequence, Nanobody or polypeptide of the invention (and in case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention, that may for instance be obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of the invention, the transformed host cell or transformed host organism may generally be kept, maintained and/or cultured under conditions such that the (desired) amino acid sequence, Nanobody or polypeptide of the invention is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium, the presence of a suitable source of food and/or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person. Again, under such conditions, the amino acid sequences of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acid sequence, Nanobody or polypeptide of the invention may (first) be generated in an immature form (as mentioned above), which may then be subjected to post-translational modification, depending on the host cell/host organism used. Also, the amino acid sequence, Nanobody or polypeptide of the invention may be glycosylated, again depending on the host cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention may then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism was cultivated, using protein isolation and/or purification techniques known per se, such as (preparative) chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g. using a specific, cleavable amino acid sequence fused with the amino acid sequence, Nanobody or polypeptide of the invention) and/or preparative immunological techniques (i.e. using antibodies against the amino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention may be formulated as a pharmaceutical preparation or compositions comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms—which may be solid, semi-solid or liquid, depending on the manner of administration—as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein.

Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one amino acid of the invention, at least one Nanobody of the invention or at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more further active substances.

Generally, the amino acid sequences, Nanobodies and polypeptides of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020,079) as well as to the standard handbooks, such as Remington's Pharmaceutical Sciences, 18^(th) Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see for example pages 252-255).

For example, the amino acid sequences, Nanobodies and polypeptides of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv's and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (i.e. transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, sterile water and aqueous buffers and solutions such as physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution; water oils; glycerol; ethanol; glycols such as propylene glycol or as well as mineral oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof. Usually, aqueous solutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and polypeptides of the invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene encoding an amino acid sequence, Nanobody or polypeptide of the invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells and can additionally be transfected with signal and stabilization sequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and polypeptides of the invention may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the amino acid sequences. Nanobodies and polypeptides of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the amino acid sequence, Nanobody or polypeptide of the invention. Their percentage in the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the amino acid sequence, Nanobody or polypeptide of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the amino acid sequences, Nanobodies and polypeptides of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the amino acid sequences, Nanobodies and polypeptides of the invention may be incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also be provided with an enteric coating that will allow the constructs of the invention to resist the gastric environment and pass into the intestines. More generally, preparations and formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the invention may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the amino acid sequences, Nanobodies and polypeptides of the invention or their salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all eases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the amino acid sequences, Nanobodies and polypeptides of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the amino acid sequences, Nanobodies and polypeptides of the invention may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the amino acid sequences, Nanobodies and polypeptides of the invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the amino acid sequences, Nanobodies and polypeptides of the invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and polypeptides of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequences, Nanobodies and polypeptides of the invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, Nanobodies and polypeptides of the invention required for use in treatment will vary not only with the particular amino acid sequence, Nanobody or polypeptide selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the amino acid sequences, Nanobodies and polypeptides of the invention varies depending on the target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By “long-term” is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E. W., ed. 4). Mack Publishing Co., Easton, Pa. The dosage can also be adjusted by the individual physician in the event of any complication.

In another aspect, the invention relates to a method for the prevention and/or treatment of at least one cancer and/or tumor, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/or treatment” not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

The invention relates to a method for the prevention and/or treatment of at least one disease or disorder that is associated with HER2, with its biological or pharmacological activity, and/or with the biological pathways or signalling in which HER2 is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be treated by modulating HER2, its biological or pharmacological activity, and/or the biological pathways or signalling in which HER2 is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, said pharmaceutically effective amount may be an amount that is sufficient to modulate HER2, its biological or pharmacological activity, and/or the biological pathways or signalling in which HER2 is involved; and/or an amount that provides a level of the amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention in the circulation that is sufficient to modulate HER2, its biological or pharmacological activity, and/or the biological pathways or signalling in which HER2 is involved.

The invention furthermore relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence of the invention, a Nanobody of the invention or a polypeptide of the invention to a patient, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder chosen from the group consisting of the diseases and disorders listed herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises administering, to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In the above methods, the amino acid sequences, Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, the amino acid sequences, Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same can for example be administered orally, intraperitoneally (e.g. intravenously, subcutaneously, intramuscularly, or via any other route of administration that circumvents the gastrointestinal tract), intranasally, transdermally, topically, by means of a suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used. The clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration, depending on the disease or disorder to be prevented or treated and other factors well known to the clinician.

The amino acid sequences, Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific amino acid sequence, Nanobody or polypeptide of the invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of one or more amino acid sequences, Nanobodies and/or polypeptides of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to administered can be determined by the clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or disorder to be treated, the potency of the specific amino acid sequence, Nanobody and polypeptide of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the amino acid sequences, Nanobodies and polypeptides of the invention will generally be administered in an amount between 1 grain and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1, 10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. Generally, some guidance on the amounts to be administered can be obtained from the amounts usually administered for comparable conventional antibodies or antibody fragments against the same target administered via essentially the same route, taking into account however differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody or polypeptide of the invention will be used. It is however within the scope of the invention to use two or more amino acid sequences. Nanobodies and/or polypeptides of the invention in combination.

The Nanobodies, amino acid sequences and polypeptides of the invention may also be used in combination with one or more further pharmaceutically active compounds or principles, i.e. as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies and polypeptides of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.

When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.

Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side-effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. The clinician will also be able, where appropriate and on a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

In another aspect, the invention relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one cancer and/or tumor; and/or for use in one or more of the methods of treatment mentioned herein.

In another aspect, the invention relates to an amino acid sequence, Nanobody or polypeptide of the invention for prevention and/or treatment of at least one cancer and/or tumor; and/or for use in one or more of the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

The invention also relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence, Nanobody or polypeptide of the invention to a patient.

More in particular, the invention relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of cancers and/or tumors, and in particular for the prevention and treatment of one or more of the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acid sequences, Nanobodies or polypeptides of the invention may also be suitably combined with one or more other active principles, such as those mentioned herein.

Finally, although the use of the Nanobodies of the invention (as defined herein) and of the polypeptides of the invention is much preferred, it will be clear that on the basis of the description herein, the skilled person will also be able to design and/or generate, in an analogous manner, other amino acid sequences and in particular (single) domain antibodies against HER2, as well as polypeptides comprising such (single) domain antibodies.

For example, it will also be clear to the skilled person that it may be possible to “graft” one or more of the CDR's mentioned above for the Nanobodies of the invention onto such (single) domain antibodies or other protein scaffolds, including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting will be clear to the skilled person and are well known in the art, see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605 522, EP 0 460 167, U.S. Pat. No. 7,054,297, Nicaise et al., Protein Science (2004), 13:1882-1891; Ewert et al., Methods, 2004 October; 34(2):184-199; Kettleborough et al., Protein Eng. 1991 October; 4(7): 773-783; O'Brien and Jones, Methods Mol. Biol. 2003:207:81-100; Skerra, J. Mol. Recognit. 2000:13:167-187, and Saerens et al., J. Mol. Biol. 2005 Sep. 23; 352(3):597-607, and the further references cited therein. For example, techniques known per se for grafting mouse or rat CDR's onto human frameworks and scaffolds can be used in an analogous manner to provide chimeric proteins comprising one or more of the CDR's of the Nanobodies of the invention and one or more human framework regions or sequences.

It should also be noted that, when the Nanobodies of the inventions contain one or more other CDR sequences than the preferred CDR sequences mentioned above, these CDR sequences can be obtained in any manner known per se, for example from Nanobodies (preferred), V_(H) domains from conventional antibodies (and in particular from human antibodies), heavy chain antibodies, conventional 4-chain antibodies (such as conventional human 4-chain antibodies) or other immunoglobulin sequences directed against HER2. Such immunoglobulin sequences directed against HER2 can be generated in any manner known per se, as will be clear to the skilled person, i.e. by immunization with HER2 or by screening a suitable library of immunoglobulin sequences with HER2, or any suitable combination thereof. Optionally, this may be followed by techniques such as random or site-directed mutagenesis and/or other techniques for affinity maturation known per se. Suitable techniques for generating such immunoglobulin sequences will be clear to the skilled person, and for example include the screening techniques reviewed by Hoogenboom, Nature Biotechnology, 23, 9, 1105-1116 (2005) Other techniques for generating immunoglobulins against a specified target include for example the Nanoclone technology (as for example described in the published US patent application 2006-0211088), so-called SLAM technology (as for example described in the European patent application 0 542 810), the use of transgenic mice expressing human immunoglobulins or the well-known hybridoma techniques (see for example Larrick et al, Biotechnology, Vol. 7, 1989, p. 934). All these techniques can be used to generate immunoglobulins against HER2, and the CDR's of such immunoglobulins can be used in the Nanobodies of the invention, i.e. as outlined above. For example, the sequence of such a CDR can be determined, synthesized and/or isolated, and inserted into the sequence of a Nanobody of the invention (e.g. so as to replace the corresponding native CDR), all using techniques known per se such as those described herein, or Nanobodies of the invention containing such CDR's (or nucleic acids encoding the same) can be synthesized de novo, again using the techniques mentioned herein.

Further uses of the amino acid sequences, Nanobodies, polypeptides, nucleic acids, genetic constructs and hosts and host cells of the invention will be clear to the skilled person based on the disclosure herein. For example, and without limitation, the amino acid sequences of the invention can be linked to a suitable carrier or solid support so as to provide a medium than can be used in a manner known per se to purify HER2 from compositions and preparations comprising the same. Derivatives of the amino acid sequences of the invention that comprise a suitable detectable label can also be used as markers to determine (qualitatively or quantitatively) the presence of HER2 in a composition or preparation or as a marker to selectively detect the presence of HER2 on the surface of a cell or tissue (for example, in combination with suitable cell sorting techniques).

The invention will now be further described by means of the following non-limiting preferred aspects, examples and figures:

PREFERRED ASPECTS

-   1. Amino acid sequence that is directed against and/or that can     specifically bind to HER2. -   2. Amino acid sequence according to aspect 1, wherein said amino     acid sequence competes with Herceptin® for binding to HER2. -   3. Amino acid sequence according to any of aspects 1 or 2, wherein     said amino acid sequence inhibits and/or blocks binding of     Herceptin® to HER -   4. Amino acid sequence according to any of aspects 1 to 3, wherein     said amino acid sequence is directed against the Herceptin® binding     site on HER2. -   5. Amino acid sequence according to any of aspects 1 to 4, wherein     said amino acid sequence specifically binds to domain IV of HER2. -   6. Amino acid sequence according to aspect 1, wherein said amino     acid sequence competes with Omnitarg for binding to HER2. -   7. Amino acid sequence according to any of aspect 1 or 7, wherein     said amino acid sequence inhibits and/or blocks binding of Omnitarg     to HER2. -   8. Amino acid sequence according to any of aspects 6 or 7, wherein     said amino acid sequence is directed against the Omnitarg binding     site on HER2. -   9. Amino acid sequence according to any of aspects 6 to 8, wherein     said amino acid sequence specifically binds to domain II of HER2. -   10. Amino acid sequence according to any of aspects 1 to 9, wherein     said amino acid competes with Herceptin® and Omnitarg for binding to     HER2. -   11. Amino acid sequence according to any of aspects 1 to 10, wherein     said amino acid sequence inhibits and/or blocks binding of     Herceptin® and Omnitarg to HER2. -   12. Amino acid sequence according to any of aspects 1 to 11, wherein     said amino acid sequence inhibits and/or blocks tumor cell     proliferation. -   13. Amino acid sequence according to any of aspects 1 to 12, wherein     said amino acid sequence inhibits, downregulates and/or blocks cell     signalling. -   14. Amino acid sequence according to any of aspects 1 to 13, wherein     said amino acid sequence induces apoptosis in tumor cells. -   15. Amino acid sequence according to any of aspects 1 to 14, wherein     said amino acid sequence inhibits and/or blocks heterodimerization     between ERBB receptors. -   16. Amino acid sequence according to any of aspects 1 to 15, wherein     said amino acid sequence inhibits and/or blocks ligand activation of     an ERbB hetero-oligomer comprising HER2 and HER3, HER4 or EGFR. -   17. Amino acid sequence according to any of aspects 1 to 16, wherein     said amino acid sequence inhibits and/or blocks tumor     vascularisation. -   18. Amino acid sequence according to any of aspects Ito 17, wherein     said amino acid sequence recruits immune effector cells such as     macrophages and monocytes to the tumor. -   19. Amino acid sequence according to any of aspects 1 to 18, wherein     said amino acid sequence inhibits and/or blocks TNF induced     signalling and/or cell proliferation. -   20. Amino acid sequence according to any of aspects 1 to 19, wherein     said amino acid sequence downregulates HER2 levels and/or     downregulates HER2-mediated signalling pathways. -   21. Amino acid sequence according to any of aspects 1 to 20, wherein     said amino acid sequence inhibits and/or blocks     metalloproteinase-mediated HER2 ectodomain shedding. -   22. Amino acid sequence according to any of aspects 1 to 21, wherein     said amino acid sequence inhibits, downregulates and/or blocks     ligand-mediated ErbB signalling. -   23. Amino acid sequence according to any of aspects 1 to 22, wherein     said amino acid sequence inhibits and/or blocks HER2 ectodomain     cleavage. -   24. Amino acid sequence according to any of aspects 1 to 23, wherein     said amino acid sequence inhibits and/or blocks Heregulin-mediated     activation of MAPK/Erk1/2. -   25. Amino acid sequence according to any of aspects 1 to 24, wherein     said amino acid sequence inhibits and/or blocks P13K/Akt signalling. -   26. Amino acid sequence according to any of aspects 1 to 25, wherein     said amino acid sequence modulates HER2 or HER2 mediated signalling     via the same mechanism of action as Herceptin®. -   27. Amino acid sequence according to any of aspects 1 to 26, wherein     said amino acid sequence modulates HER2 or HER2 mediated signalling     via the same mechanism of action as Omnitarg. -   28. Amino acid sequence according to any of aspects 1 to 27, that is     in essentially isolated form. -   29. Amino acid sequence according to aspect 1 or 28, for     administration to a subject, wherein said amino acid sequence does     not naturally occur in said subject. -   30. Amino acid sequence according to any of aspects 1 to 29, that     can specifically bind to HER2 with a dissociation constant (K_(D))     of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²     moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre. -   31. Amino acid sequence according to any of aspects 1 to 30, that     can specifically bind to HER2 with a rate of association     (k_(on)-rate) of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹ preferably     between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹     s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹. -   32. Amino acid sequence according to any of aspects 1 to 31, that     can specifically bind to HER2 with a rate of dissociation (k_(off)     rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹, preferably between 10⁻² s⁻¹ and     10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as     between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹. -   33. Amino acid sequence according to any of aspects 1 to 32, that     can specifically bind to HER2 with an affinity less than 500 nM,     preferably less than 200 nM, more preferably less than 10 nM, such     as less than 500 μM. -   34. Amino acid sequence according to any of aspects 1 to 33, that is     a naturally occurring amino acid sequence (from any suitable     species) or a synthetic or semi-synthetic amino acid sequence. -   35. Amino acid sequence according to any of aspects 1 to 34, that     comprises an immunoglobulin fold or that under suitable conditions     is capable of forming an immunoglobulin fold. -   36. Amino acid sequence according to any of aspects 1 to 35, that     essentially consists of 4 framework regions (FR1 to FR4     respectively) and 3 complementarity determining regions (CDR1 to     CDR3 respectively). -   37. Amino acid sequence according to any of aspects 1 to 36, that is     an immunoglobulin sequence. -   38. Amino acid sequence according to any of aspects 1 to 37, that is     a naturally occurring immunoglobulin sequence (from any suitable     species) or a synthetic or semi-synthetic immunoglobulin sequence. -   39. Amino acid sequence according to any of aspects 1 to 38, that is     a humanized immunoglobulin sequence, a camelized immunoglobulin     sequence or an immunoglobulin sequence that has been obtained by     techniques such as affinity maturation. -   40. Amino acid sequence according to any of aspects 1 to 39, that     essentially consists of a light chain variable domain sequence (e.g.     a V_(L)-sequence); or of a heavy chain variable domain sequence     (e.g. a V_(H)-sequence). -   41. Amino acid sequence according to any of aspects 1 to 40, that     essentially consists of a heavy chain variable domain sequence that     is derived from a conventional four-chain antibody or that     essentially consist of a heavy chain variable domain sequence that     is derived from heavy chain antibody. -   42. Amino acid sequence according to any of aspects 1 to 41, that     essentially consists of a domain antibody (or an amino acid sequence     that is suitable for use as a domain antibody), of a single domain     antibody (or an amino acid sequence that is suitable for use as a     single domain antibody), of a “dAb” (or an amino acid sequence that     is suitable for use as a dAb) or of a Nanobody® (including but not     limited to a V_(HH) sequence). -   43. Amino acid sequence according to any of aspects 1 to 42, that     essentially consists of a Nanobody®. -   44. Amino acid sequence according to any of aspects 1 to 43, that     essentially consists of a Nanobody® that     -   i) has at least 80% amino acid identity with at least one of the         amino acid sequences of SEQ ID NO's: 1 to 22, in which for the         purposes of determining the degree of amino acid identity, the         amino acid residues that form the CDR sequences are disregarded;     -   and in which:     -   ii) preferably one or more of the amino acid residues at         positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according         to the Kabat numbering are chosen from the Hallmark residues         mentioned in Table A-3. -   45. Amino acid sequence according to any of aspects 1 to 44, that     essentially consists of a Nanobody® that     -   i) has at least 80% amino acid identity with at least one of the         amino acid sequences of SEQ ID NO's: 2051-2325, in which for the         purposes of determining the degree of amino acid identity, the         amino acid residues that form the CDR sequences are disregarded;     -   and in which:     -   ii) preferably one or more of the amino acid residues at         positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according         to the Kabat numbering are chosen from the Hallmark residues         mentioned in Table A-3. -   46. Amino acid sequence according to any of aspects 1 to 45, that     essentially consists of a humanized Nanobody®. -   47. Amino acid sequence according to any of aspects 1 to 46, that in     addition to the at least one binding site for binding against HER2,     contains one or more further binding sites for binding against other     antigens, proteins or targets. -   48. Amino acid sequence directed against HER2, that comprises one or     more stretches of amino acid residues chosen from the group     consisting of:     -   a) the amino acid sequences of SEQ ID NO's: 401-675;     -   b) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 401-675;     -   c) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 401-675;     -   d) the amino acid sequences of SEQ ID NO's: 951-1225;     -   e) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 951-1225;     -   f) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 951-1225;     -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;     -   h) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 1501-1775;     -   i) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 1501-1775;     -   or any suitable combination thereof. -   49. Amino acid sequence according to aspect 48, in which at least     one of said stretches of amino acid residues forms part of the     antigen binding site for binding against HER2. -   50. Amino acid sequence according to any of aspects 48 or 49, that     comprises two or more stretches of amino acid residues chosen from     the group consisting of:     -   a) the amino acid sequences of SEQ ID NO's: 401-675;     -   b) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 401-675;     -   c) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 401-675;     -   d) the amino acid sequences of SEQ ID NO's: 951-1225;     -   e) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 951-1225;     -   f) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 951-1225;     -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;     -   h) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 1501-1775;     -   i) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 1501-1775;     -   such that (i) when the first stretch of amino acid residues         corresponds to one of the amino acid sequences according to         a), b) or c), the second stretch of amino acid residues         corresponds to one of the amino acid sequences according to d),         e), f), g), f) or i); (ii) when the first stretch of amino acid         residues corresponds to one of the amino acid sequences         according to d), e) or f), the second stretch of amino acid         residues corresponds to one of the amino acid sequences         according to a), b), c), g), h) or i); or (iii) when the first         stretch of amino acid residues corresponds to one of the amino         acid sequences according to g), h) or i), the second stretch of         amino acid residues corresponds to one of the amino acid         sequences according to a), b), c), d), e) or f). -   51. Amino acid sequence according to aspect 50, in which the at     least two stretches of amino acid residues forms part of the antigen     binding site for binding against HER2. -   52. Amino acid sequence according to any of aspects 48 to 51, that     comprises three or more stretches of amino acid residues, in which     the first stretch of amino acid residues is chosen from the group     consisting of:     -   a) the amino acid sequences of SEQ ID NO's: 401-675;     -   b) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 401-675;     -   c) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 401-675;     -   the second stretch of amino acid residues is chosen from the         group consisting of:     -   d) the amino acid sequences of SEQ ID NO's: 951-1225;     -   e) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 951-1225;     -   f) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 951-1225;     -   and the third stretch of amino acid residues is chosen from the         group consisting of:     -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;     -   h) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 1501-1775;     -   i) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 1501-1775. -   53. Amino acid sequence according to aspect 52, in which the at     least three stretches of amino acid residues forms part of the     antigen binding site for binding against HER2. -   54. Amino acid sequence according to any of aspects 48 to 53, in     which the CDR sequences of said amino acid sequence have at least     70% amino acid identity, preferably at least 80% amino acid     identity, more preferably at least 90% amino acid identity, such as     95% amino acid identity or more or even essentially 100% amino acid     identity with the CDR sequences of at least one of the amino acid     sequences of SEQ ID NO's: 2051-2325. -   55. Amino acid sequence directed against HER2 that cross-blocks the     binding of at least one of the amino acid sequences according to any     of aspects 48 to 54 to HER2. -   56. Amino acid sequence directed against HER2 that is cross-blocked     from binding to HER2 by at least one of the amino acid sequences     according to any of aspects 48 to 54. -   57. Amino acid sequence according to any of aspects 55 or 56,     wherein the ability of said amino acid sequence to cross-block or to     be cross-blocked is detected in a Biacore assay. -   58. Amino acid sequence according to any of aspects 55 to 57,     wherein the ability of said amino acid sequence to cross-block or to     be cross-blocked is detected in an ELISA assay. -   59. Amino acid sequence according to any of aspects 58 to 58, that     is in essentially isolated form. -   60. Amino acid sequence according to any of aspects 48 to 59, for     administration to a subject, wherein said amino acid sequence does     not naturally occur in said subject. -   61. Amino acid sequence according to any of aspects 48 to 60, that     can specifically bind to HER2 with a dissociation constant (K_(D))     of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²     moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre. -   62. Amino acid sequence according to any of aspects 48 to 61, that     can specifically bind to HER2 with a rate of association     (k_(on)-rate) of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,     preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably     between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and     10⁷ M⁻¹ s⁻¹. -   63. Amino acid sequence according to any of aspects 48 to 62, that     can specifically bind to HER2 with a rate of dissociation (k_(off)     rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and     10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as     between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹. -   64. Amino acid sequence according to any of aspects 48 to 63, that     can specifically bind to HER2 with an affinity less than 500 nM,     preferably less than 200 nM, more preferably less than 10 nM, such     as less than 500 μM. -   65. Amino acid sequence according to any of aspects 48 to 64, that     is a naturally occurring amino acid sequence (from any suitable     species) or a synthetic or semi-synthetic amino acid sequence. -   66. Amino acid sequence according to any of aspects 48 to 65, that     comprises an immunoglobulin fold or that under suitable conditions     is capable of forming an immunoglobulin fold. -   67. Amino acid sequence according to any of aspects 48 to 66, that     is an immunoglobulin sequence. -   68. Amino acid sequence according to any of aspects 48 to 67, that     is a naturally occurring immunoglobulin sequence (from any suitable     species) or a synthetic or semi-synthetic immunoglobulin sequence. -   69. Amino acid sequence according to any of aspects 48 to 68, that     is a humanized immunoglobulin sequence, a camelized immunoglobulin     sequence or an immunoglobulin sequence that has been obtained by     techniques such as affinity maturation. -   70. Amino acid sequence according to any of aspects 48 to 69, that     essentially consists of a light chain variable domain sequence (e.g.     a V_(L)-sequence); or of a heavy chain variable domain sequence     (e.g. a V_(H)-sequence). -   71. Amino acid sequence according to any of aspects 48 to 70, that     essentially consists of a heavy chain variable domain sequence that     is derived from a conventional four-chain antibody or that     essentially consist of a heavy chain variable domain sequence that     is derived from heavy chain antibody. -   72. Amino acid sequence according to any of aspects 48 to 71, that     essentially consists of a domain antibody (or an amino acid sequence     that is suitable for use as a domain antibody), of a single domain     antibody (or an amino acid sequence that is suitable for use as a     single domain antibody), of a “dAb” (or an amino acid sequence that     is suitable for use as a dAb) or of a Nanobody® (including but not     limited to a V_(HH) sequence). -   73. Amino acid sequence according to any of aspects 48 to 72, that     essentially consists of a Nanobody®. -   74. Amino acid sequence according to any of aspects 48 to 73, that     essentially consists of a Nanobody® that     -   i) has at least 80% amino acid identity with at least one of the         amino acid sequences of SEQ ID NO's: 1 to 22, in which for the         purposes of determining the degree of amino acid identity, the         amino acid residues that form the CDR sequences are disregarded;     -   and in which:     -   ii) preferably one or more of the amino acid residues at         positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according         to the Kabat numbering are chosen from the Hallmark residues         mentioned in Table A-3. -   75. Amino acid sequence according to any of aspects 48 to 74, that     essentially consists of a Nanobody® that     -   i) has at least 80% amino acid identity with at least one of the         amino acid sequences of SEQ ID NO's: 2051-2325, in which for the         purposes of determining the degree of amino acid identity, the         amino acid residues that form the CDR sequences are disregarded;     -   and in which:     -   ii) preferably one or more of the amino acid residues at         positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according         to the Kabat numbering are chosen from the Hallmark residues         mentioned in Table A-3. -   76. Amino acid sequence according to any of aspects 48 to 75, that     essentially consists of a humanized Nanobody®. -   77. Amino acid sequence according to any of aspects 1 to 76, that in     addition to the at least one binding site for binding formed by the     CDR sequences, contains one or more further binding sites for     binding against other antigens, proteins or targets. -   78. Amino acid sequence that essentially consists of 4 framework     regions (FR1 to FR4, respectively) and 3 complementarity determining     regions (CDR1 to CDR3, respectively), in which:     -   CDR1 is chosen from the group consisting of:     -   a) the amino acid sequences of SEQ ID NO's: 401-675;     -   b) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 401-675;     -   c) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 401-675;     -   and/or     -   CDR2 is chosen from the group consisting of:     -   d) the amino acid sequences of SEQ ID NO's: 951-1225;     -   e) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 951-1225;     -   f) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 951-1225;     -   and/or     -   CDR3 is chosen from the group consisting of:     -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;     -   h) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 1501-1775;     -   i) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 1501-1775. -   79. Amino acid sequence that essentially consists of 4 framework     regions (FR1 to FR4, respectively) and 3 complementarity determining     regions (CDR1 to CDR3, respectively), in which:     -   CDR1 is chosen from the group consisting of:     -   a) the amino acid sequences of SEQ ID NO's: 401-675;     -   b) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 401-675;     -   c) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 401-675;     -   and     -   CDR2 is chosen from the group consisting of:     -   d) the amino acid sequences of SEQ ID NO's: 951-1225;     -   e) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 951-1225;     -   f) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 951-1225;     -   and     -   CDR3 is chosen from the group consisting of:     -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;     -   h) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 1501-1775;     -   i) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 1501-1775]. -   80. Amino acid sequence according to any of aspects 78 to 79, in     which the CDR sequences of said amino acid sequence have at least     70% amino acid identity, preferably at least 80% amino acid     identity, more preferably at least 90% amino acid identity, such as     95% amino acid identity or more or even essentially 100% amino acid     identity with the CDR sequences of at least one of the amino acid     sequences of SEQ ID NO's: 2051-2325. -   81. Amino acid sequence directed against HER2 that cross-blocks the     binding of at least one of the amino acid sequences according to any     of aspects 78 to 80 to HER2. -   82. Amino acid sequence directed against HER2 that is cross-blocked     from binding to HER2 by at least one of the amino acid sequences     according to any of aspects 78 to 80. -   83. Amino acid sequence according to any of aspects 81 or 82,     wherein the ability of said amino acid sequence to cross-block or to     be cross-blocked is detected in a Biacore assay. -   84. Amino acid sequence according to any of aspects 81 to 83,     wherein the ability of said amino acid sequence to cross-block or to     be cross-blocked is detected in an ELISA assay. -   85. Amino acid sequence according to any of aspects 78 to 84, that     is in essentially isolated form. -   86. Amino acid sequence according to any of aspects 78 to 85, for     administration to a subject, wherein said amino acid sequence does     not naturally occur in said subject. -   87. Amino acid sequence according to any of aspects 78 to 86, that     can specifically bind to HER2 with a dissociation constant (K_(D))     of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²     moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre. -   88. Amino acid sequence according to any of aspects 78 to 87, that     can specifically bind to HER2 with a rate of association     (k_(on)-rate) of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,     preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably     between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and     10⁷ M⁻¹ s⁻¹. -   89. Amino acid sequence according to any of aspects 78 to 88, that     can specifically bind to HER2 with a rate of dissociation (k_(off)     rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and     10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as     between 10⁻⁴ s⁻¹ and 10⁻⁶ S⁻¹. -   90. Amino acid sequence according to any of aspects 78 to 89, that     can specifically bind to HER2 with an affinity less than 500 nM,     preferably less than 200 nM, more preferably less than 10 nM, such     as less than 500 μM. -   91. Amino acid sequence according to any of aspects 78 to 90, that     is a naturally occurring amino acid sequence (from any suitable     species) or a synthetic or semi-synthetic amino acid sequence. -   92. Amino acid sequence according to any of aspects 78 to 91, that     comprises an immunoglobulin fold or that under suitable conditions     is capable of forming an immunoglobulin fold. -   93. Amino acid sequence according to any of aspects 78 to 92, that     is an immunoglobulin sequence. -   94. Amino acid sequence according to any of aspects 78 to 93, that     is a naturally occurring immunoglobulin sequence (from any suitable     species) or a synthetic or semi-synthetic immunoglobulin sequence. -   95. Amino acid sequence according to any of aspects 78 to 94, that     is a humanized immunoglobulin sequence, a camelized immunoglobulin     sequence or an immunoglobulin sequence that has been obtained by     techniques such as affinity maturation. -   96. Amino acid sequence according to any of aspects 78 to 95, that     essentially consists of a light chain variable domain sequence (e.g.     a V_(L)-sequence); or of a heavy chain variable domain sequence     (e.g. a V_(H)-sequence). -   97, Amino acid sequence according to any of aspects 78 to 96, that     essentially consists of a heavy chain variable domain sequence that     is derived from a conventional four-chain antibody or that     essentially consist of a heavy chain variable domain sequence that     is derived from heavy chain antibody. -   98. Amino acid sequence according to any of aspects 78 to 97, that     essentially consists of a domain antibody (or an amino acid sequence     that is suitable for use as a domain antibody), of a single domain     antibody (or an amino acid sequence that is suitable for use as a     single domain antibody), of a “dAb” (or an amino acid sequence that     is suitable for use as a dAb) or of a Nanobody® (including but not     limited to a V_(HH) sequence). -   99. Amino acid sequence according to any of aspects 78 to 98, that     essentially consists of a Nanobody®. -   100. Amino acid sequence according to any of aspects 78 to 99, that     essentially consists of a Nanobody® that     -   i) has at least 80% amino acid identity with at least one of the         amino acid sequences of SEQ ID NO's: 1 to 22, in which for the         purposes of determining the degree of amino acid identity, the         amino acid residues that form the CDR sequences are disregarded;     -   and in which:     -   ii) preferably one or more of the amino acid residues at         positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according         to the Kabat numbering are chosen from the Hallmark residues         mentioned in Table A-3. -   101. Amino acid sequence according to any of aspects 78 to 100, that     essentially consists of a Nanobody® that     -   i) has at least 80% amino acid identity with at least one of the         amino acid sequences of SEQ ID NO's: 2051-2325, in which for the         purposes of determining the degree of amino acid identity, the         amino acid residues that form the CDR sequences are disregarded;     -   and in which:     -   ii) preferably one or more of the amino acid residues at         positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according         to the Kabat numbering are chosen from the Hallmark residues         mentioned in Table A-3. -   102. Amino acid sequence according to any of aspects 78 to 101, that     essentially consists of a humanized Nanobody®. -   103. Amino acid sequence according to any of aspects 78 to 102, that     in addition to the at least one binding site for binding formed by     the CDR sequences, contains one or more further binding sites for     binding against other antigens, proteins or targets. -   104. Nanobody that is directed against and/or that can specifically     bind to HER2. -   105. Nanobody according to aspect 104, wherein said Nanobody     competes with Herceptin® for binding to HER2. -   106. Nanobody according to any of aspects 104 or 105, wherein said     Nanobody inhibits and/or blocks binding of Herceptin® to HER2. -   107. Nanobody according to any of aspects 104 to 106, wherein said     Nanobody is directed against the Herceptin® binding site on HER2. -   108. Nanobody according to any of aspects 104 to 107, wherein said     Nanobody specifically binds to domain IV of HER2. -   109. Nanobody according to aspect 104, wherein said Nanobody     competes with Omnitarg for binding to HER2. -   110. Nanobody according to any of aspects 104 or 109, wherein said     Nanobody inhibits and/or blocks binding of Omnitarg to HER2. -   111. Nanobody according to any of aspects 109 or 110, wherein said     Nanobody is directed against the Omnitarg binding site on HER2. -   112. Nanobody according to any of aspects 109 to 111, wherein said     Nanobody specifically binds to domain II of HER2. -   113. Nanobody according to any of aspects 104 to 112, wherein said     Nanobody competes with Herceptin® and Omnitarg for binding to HER2. -   114. Nanobody according to any of aspects 104 to 113, wherein said     Nanobody inhibits and/or blocks binding of Herceptin® and Omnitarg     to HER2. -   115. Nanobody according to any of aspects 104 to 114, wherein said     Nanobody inhibits and/or blocks tumor cell proliferation. -   116. Nanobody according to any of aspects 104 to 115, wherein said     Nanobody inhibits, downregulates and/or blocks cell signalling. -   117. Nanobody according to any of aspects 104 to 116, wherein said     Nanobody induces apoptosis in tumor cells. -   118. Nanobody according to any of aspects 104 to 117, wherein said     Nanobody inhibits and/or blocks heterodimerization between ERBB     receptors. -   119. Nanobody according to any of aspects 104 to 118, wherein said     Nanobody inhibits and/or blocks ligand activation of an ERbB     hetero-oligomer comprising HER2 and HER3, HER4 or EGFR. -   120. Nanobody according to any of aspects 104 to 119, wherein said     Nanobody inhibits and/or blocks tumor vascularisation. -   121. Nanobody according to any of aspects 104 to 120, wherein said     Nanobody recruits immune effector cells such as macrophages and     monocytes to the tumor. -   122. Nanobody according to any of aspects 104 to 121, wherein said     Nanobody inhibits and/or blocks TNF induced signalling and/or cell     proliferation. -   123. Nanobody according to any of aspects 104 to 122, wherein said     Nanobody downregulates HER2 levels and/or downregulates     HER2-mediated signalling pathways. -   124. Nanobody according to any of aspects 104 to 123, wherein said     Nanobody inhibits and/or blocks metalloproteinase-mediated HER2     ectodomain shedding. -   125. Nanobody according to any of aspects 104 to 124, wherein said     Nanobody downregulates and/or blocks ligand-mediated ErbB     signalling. -   126. Nanobody according to any of aspects 104 to 125, wherein said     Nanobody inhibits and/or blocks HER2 ectodomain cleavage. -   127. Nanobody according to any of aspects 104 to 126, wherein said     Nanobody inhibits and/or blocks Heregulin-mediated activation of     MAPK/Erk1/2. -   128. Nanobody according to any of aspects 104 to 127, wherein said     Nanobody inhibits and/or blocks PI3K/Akt signalling. -   129. Nanobody according to any of aspects 104 to 128, wherein said     Nanobody modulates HER2 or HER2 mediated signalling via the same     mechanism of action as Herceptin®. -   130. Nanobody according to any of aspects 104 to 129, wherein said     Nanobody modulates HER2 or HER2 mediated signalling via the same     mechanism of action as Omnitarg. -   131. Nanobody according to any of aspects 104 to 130, that is in     essentially isolated form. -   132. Nanobody according to any of aspects 104 to 131, that can     specifically bind to HER2 with a dissociation constant (K_(D)) of     10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²     moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre. -   133. Nanobody according to any of aspects 104 to 132, that can     specifically bind to HER2 with a rate of association (k_(on)-rate)     of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹, preferably between 10³     M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷     M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹. -   134. Nanobody according to any of aspects 104 to 133, that can     specifically bind to HER2 with a rate of dissociation (k_(off) rate)     between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,     more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴     s⁻¹ and 10⁻⁶ s⁻¹. -   135. Nanobody according to any of aspects 104 to 134, that can     specifically bind to HER2 with an affinity less than 500 nM,     preferably less than 200 nM, more preferably less than 10 nM, such     as less than 500 μM. -   136. Nanobody according to any of aspects 104 to 135, that is a     naturally occurring Nanobody (from any suitable species) or a     synthetic or semi-synthetic Nanobody. -   137. Nanobody according to any of aspects 104 to 136, that is a     V_(HH) sequence, a partially humanized V_(HH) sequence, a fully     humanized V_(HH) sequence, a camelized heavy chain variable domain     or a Nanobody that has been obtained by techniques such as affinity     maturation. -   138. Nanobody according to any of aspects 104 to 137, that     -   i) has at least 80% amino acid identity with at least one of the         amino acid sequences of SEQ ID NO's: 1 to 22, in which for the         purposes of determining the degree of amino acid identity, the         amino acid residues that form the CDR sequences are disregarded;     -   and in which:     -   ii) preferably one or more of the amino acid residues at         positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according         to the Kabat numbering are chosen from the Hallmark residues         mentioned in Table A-3. -   139. Nanobody according to any of aspects 104 to 138, that     -   i) has at least 80% amino acid identity with at least one of the         amino acid sequences of SEQ ID NO's: 2051-2325, in which for the         purposes of determining the degree of amino acid identity, the         amino acid residues that form the CDR sequences are disregarded;     -   and in which:     -   ii) preferably one or more of the amino acid residues at         positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according         to the Kabat numbering are chosen from the Hallmark residues         mentioned in Table A-3. -   140. Nanobody according to any of aspects 104 to 139, in which:     -   CDR1 is chosen from the group consisting of:     -   a) the amino acid sequences of SEQ ID NO's: 401-675;     -   b) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 401-675;     -   c) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 401-675;     -   and/or     -   CDR2 is chosen from the group consisting of:     -   d) the amino acid sequences of SEQ ID NO's: 951-1225;     -   e) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 951-1225;     -   f) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 951-1225;     -   and/or     -   CDR3 is chosen from the group consisting of:     -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;     -   h) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 1501-1775;     -   i) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 1501-1775. -   141. Nanobody according to any of aspects 104 to 140, in which:     -   CDR1 is chosen from the group consisting of:     -   a) the amino acid sequences of SEQ ID NO's: 401-675;     -   b) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 401-675;     -   c) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 401-675;     -   and     -   CDR2 is chosen from the group consisting of:     -   d) the amino acid sequences of SEQ ID NO's: 951-1225;     -   e) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 951-1225;     -   f) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 951-1225;     -   and     -   CDR3 is chosen from the group consisting of:     -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;     -   h) amino acid sequences that have at least 80% amino acid         identity with at least one of the amino acid sequences of SEQ ID         NO's: 1501-1775;     -   i) amino acid sequences that have 3, 2, or 1 amino acid         difference with at least one of the amino acid sequences of SEQ         ID NO's: 1501-1775. -   142. Nanobody according to any of aspects 104 to 141, in which the     CDR sequences have at least 70% amino acid identity, preferably at     least 80% amino acid identity, more preferably at least 90% amino     acid identity, such as 95% amino acid identity or more or even     essentially 100% amino acid identity with the CDR sequences of at     least one of the amino acid sequences of SEQ ID NO's: 2051-2325. -   143. Nanobody according to any of aspects 104 to 1.42, which is a     partially humanized Nanobody. -   144. Nanobody according to any of aspects 104 to 143, which is a     fully humanized Nanobody. -   145. Nanobody according to any of aspects 104 to 144, that is chosen     from the group consisting of SEQ ID NO's: 2051-2325 or from the     group consisting of amino acid sequences that have more than 80%,     preferably more than 90%, more preferably more than 95%, such as 99%     or more sequence identity (as defined herein) with at least one of     the amino acid sequences of SEQ ID NO's: 2051-2325. -   146. Nanobody according to any of aspects 104 to 145, that is chosen     from the group consisting of SEQ ID NO's: 2051-2325. -   147. Nanobody directed against HER2 that cross-blocks the binding of     at least one of the Nanobodies according to any of aspects 140 to     146 to HER2. -   148. Nanobody directed against HER2 that is cross-blocked from     binding to HER2 by at least one of the Nanobodies according to any     of aspects 140 to 146. -   149. Nanobody according to any of aspects 147 or 148, wherein the     ability of said Nanobody to cross-block or to be cross-blocked is     detected in a Biacore assay. -   150. Nanobody according to any of aspects 147 to 149, wherein the     ability of said Nanobody to cross-block or to be cross-blocked is     detected in an ELISA assay. -   151. Compound or construct, that comprises or essentially consists     of one or more amino acid sequences according to any of aspects 1 to     103 and/or one or more Nanobodies according to any of aspects 104 to     150, and optionally further comprises one or more other groups,     residues, moieties or binding units, optionally linked via one or     more linkers. -   152. Compound or construct according to aspect 151, in which said     one or more other groups, residues, moieties or binding units are     amino acid sequences. -   153. Compound or construct according to aspect 151, in which said     one or more linkers, if present, are one or more amino acid     sequences. -   154. Compound or construct according to any of aspects 151 to 153,     in which said one or more other groups, residues, moieties or     binding units are immunoglobulin sequences. -   155. Compound or construct according to any of aspects 151 to 154,     in which said one or more other groups, residues, moieties or     binding units are chosen from the group consisting of domain     antibodies, amino acid sequences that are suitable for use as a     domain antibody, single domain antibodies, amino acid sequences that     are suitable for use as a single domain antibody, “dAb”'s, amino     acid sequences that are suitable for use as a dAb, or Nanobodies. -   156. Compound or construct according to any of aspect 151 to 155, in     which said one or more amino acid sequences of the invention are     immunoglobulin sequences. -   157. Compound or construct according to any of aspects 151 to 156,     in which said one or more amino acid sequences of the invention are     chosen from the group consisting of domain antibodies, amino acid     sequences that are suitable for use as a domain antibody, single     domain antibodies, amino acid sequences that are suitable for use as     a single domain antibody, “dAb”'s, amino acid sequences that are     suitable for use as a dAb, or Nanobodies. -   158. Compound or construct, that comprises or essentially consists     of one or more Nanobodies according to any of aspects 104 to 150 and     in which said one or more other groups, residues, moieties or     binding units are Nanobodies. -   159. Compound or construct according to any of aspects 151 to 158,     which is a multivalent construct. -   160. Compound or construct according to aspect 159, that is chosen     from the group consisting of SEQ ID NO's: 2326-2330 or from the     group consisting of from amino acid sequences that have more than     80%, preferably more than 90%, more preferably more than 95%, such     as 99% or more sequence identity (as defined herein) with at least     one of the amino acid sequences of SEQ ID NO's: 2326-2330. -   161. Compound or construct according to any of aspects 151 to 160,     which is a multiparatopic construct. -   162. Compound or construct according to aspect 161, which is a     biparatopic or triparatopic construct. -   163. Compound or construct according to aspect 162, which comprises     at least one amino acid sequence directed against a first antigenic     determinant, epitope, part or domain of HER-2 and at least one amino     acid sequence directed against a second antigenic determinant,     epitope, part or domain of HER-2 different from the first antigenic     determinant, epitope, part or domain -   164. Biparatopic compound or construct according to aspect 163,     which is capable of simultaneously binding to said first antigenic     determinant, epitope, part or domain of HER2 and to said second     antigenic determinant, epitope, part or domain of HER2. -   165. Compound or construct according to any of aspects 151 to 164,     which combines two or more different modes of action each mediated     by one of its binding units, wherein each binding unit binds at a     different binding site of HER2. -   166. Compound or construct according to any of aspects 151 to 165,     which modulates, inhibits, downregulate and/or blocks two different     cell signaling pathways, each mediated by one of its binding units,     wherein each binding unit binds at a different binding site of HER2. -   167. Compound or construct according to aspect 151 to 166, wherein     said compound or construct competes with Herceptin® for binding to     HER2. -   168. Compound or construct according to any of aspects 151 to 167,     wherein said compound or construct inhibits and/or blocks binding of     Herceptin® to HER2. -   169. Compound or construct according to any of aspects 151 to 168,     wherein said compound or construct is directed against the     Herceptin® binding site on HER2. -   170. Compound or construct according to any of aspects 151 to 169,     wherein said compound or construct specifically binds to domain IV     of HER2. -   171. Compound or construct according to any of aspects 151 to 170,     wherein said compound or construct recruits immune effector cells     such as macrophages and monocytes to the tumor. -   172. Compound or construct according to any of aspects 151 to 171,     wherein said compound or construct downregulates HER2 levels and/or     downregulates HER2-mediated signalling pathways. -   173. Compound or construct according to any of aspects 151 to 172,     wherein said compound or construct inhibits and/or blocks     metalloproteinase-mediated. HER2 ectodomain shedding. -   174. Compound or construct according to any of aspects 151 to 173,     wherein said compound or construct modulates HER2 or HER2 mediated     signalling via the same mechanism of action as Herceptin®. -   175. Compound or construct according to aspect 151 to 174, wherein     said compound or construct competes with Omnitarg for binding to     HER2. -   176. Compound or construct according to any of aspects 151 to 175,     wherein said compound or construct inhibits and/or blocks binding of     Omnitarg to HER2. -   177. Compound or construct according to any of aspects 151 to 176,     wherein said compound or construct is directed against the Omnitarg     binding site on HER2. -   178. Compound or construct according to any of aspects 151 to 177,     wherein said compound or construct specifically binds to domain II     of HER2. -   179. Compound or construct according to any of aspects 151 to 178,     wherein said compound or construct inhibits and/or blocks ligand     activation of an ERbB hetero-oligomer comprising HER2 and HER3, HER4     or EGFR. -   180. Compound or construct according to any of aspects 151 to 179,     wherein said compound or construct is directed against the     Herceptin® binding site on HER2 and against the Omnitarg binding     site on HER2. -   181. Compound or construct according to any of aspects 151 to 180,     wherein said compound or construct modulates HER2 or HER2 mediated     signalling via the same mechanism of action as Omnitarg. -   182. Compound or construct according to any of aspects 151 to 181,     wherein said compound or construct competes with Herceptin® and     Omnitarg for binding to HER2. -   183. Compound or construct according to any of aspects 151 to 182,     wherein said compound or construct inhibits and/or blocks binding of     Herceptin® and Omnitarg to HER2. -   184. Compound or construct according to any of aspects 151 to 183,     which specifically binds the Omnitarg binding site on HER2 and to     the Herceptin® binding site on HER2. -   185. Compound or construct according to any of aspects 151 to 184,     which specifically binds to domain IV and domain II of HER2. -   186. Biparatopic compound or construct according to any of aspects     151 to 185, which can simultaneously bind the Omnitarg binding site     on HER2 and to the Herceptin® binding site on HER2. -   187. Compound or construct according to any of aspects 151 to 186,     which down-regulates HER2 levels while at the same time inhibiting     and/or blocking heterodimerization between ERBB receptors. -   188. Compound or construct according to any of aspects 151 to 187,     which combines the mode of action of Herceptin® and Omnitarg. -   189. Compound or construct according to any of aspects 151 to 188,     wherein said compound or construct inhibits and/or blocks tumor cell     proliferation. -   190. Compound or construct according to any of aspects 151 to 189,     wherein said compound or construct inhibits, downregulates and/or     blocks cell signalling. -   191. Compound or construct according to any of aspects 151 to 190,     wherein said compound or construct induces apoptosis in tumor cells. -   192. Compound or construct according to any of aspects 151 to 191,     wherein said compound or construct inhibits and/or blocks     heterodimerization between ERBB receptors. -   193. Compound or construct according to any of aspects 151 to 192,     wherein said compound or construct inhibits and/or blocks tumor     vascularisation. -   194. Compound or construct according to any of aspects 151 to 193,     wherein said compound or construct inhibits and/or blocks TNF     induced signalling and/or cell proliferation. -   195. Compound or construct according to any of aspects 151 to 194,     wherein said compound or construct inhibits, downregulates and/or     blocks ligand-mediated ErbB signalling. -   196. Compound or construct according to any of aspects 151 to 195,     wherein said compound or construct inhibits and/or blocks HER2     ectodomain cleavage. -   197. Compound or construct according to any of aspects 151 to 196,     wherein said compound or construct inhibits and/or blocks     Heregulin-mediated activation of MAPK/Erk1/2. -   198. Compound or construct according to any of aspects 151 to 197,     wherein said compound or construct inhibits and/or blocks PI3K/Akt -   199. Compound or construct according to any of aspects 151 to 198,     wherein said compound or construct is linked to an Fc portion. -   200. Compound or construct that comprises an Fc portion that is     linked, optionally via a suitable linker or hinge region, to a pair     of first amino acid sequences according to any of aspects 1 to 103     or Nanobodies according to any of aspects 104 to 150, which are     linked, optionally via a suitable linker, to a pair of second amino     acid sequences according to any of aspects 1 to 103 or Nanobodies     according to any of aspects 104 to 150, wherein:     -   both of the first amino acid sequences according to any of         aspects 1 to 103 or Nanobodies according to any of aspects 104         to 150 are directed against a first epitope, antigenic         determinant, part, domain or subunit on HER2; and     -   both of the second amino acid sequences according to any of         aspects 1 to 103 or Nanobodies according to any of aspects 104         to 150 are directed against a second epitope, antigenic         determinant, part, domain or subunit on HER2 which is different         from said first epitope, antigenic determinant, part, domain or         subunit. -   201. Compound or construct according to any of aspects 199 or 200,     wherein the first amino acid sequences according to any of aspects 1     to 103 or Nanobodies according to any of aspects 104 to 150 are     directed against the Herceptin® binding site on HER-2 and/or are     amino acid sequences or Nanobodies that are capable of competing     with Herceptin® for binding to HER-2. -   202. Compound or construct according to any of aspects 199 to 201,     wherein the first amino acid sequences according to any of aspects 1     to 103 or Nanobodies according to any of aspects 104 to 150 are     directed against the Omnitarg® binding site on HER-2 and/or are     amino acid sequences or Nanobodies that are capable of competing     with Omnitarg® for binding to HER-2. -   203. Compound or construct according to any of aspects 199 to 202,     wherein the first amino acid sequences according to any of aspects 1     to 103 or Nanobodies according to any of aspects 104 to 150 are     directed against the Herceptin® binding site on HER-2 and/or are     amino acid sequences or Nanobodies that are capable of competing     with Herceptin® for binding to HER-2 and the second amino acid     sequences according to any of aspects 1 to 103 or Nanobodies     according to any of aspects 104 to 150 are directed against the     Omnitarg® binding site on HER-2 and/or are amino acid sequences or     Nanobodies that are capable of competing with Omnitarg® for binding     to HER-2 (or visa versa). -   204. Compound or construct according to any of aspects 151 to 203,     that comprises or that is chosen from the group consisting of SEQ ID     NO's: 2336-2390 or from the group consisting of from amino acid     sequences that have more than 80%, preferably more than 90%, more     preferably more than 95%, such as 99% or more sequence identity (as     defined herein) with at least one of the amino acid sequences of SEQ     ID NO's: 2336-2390. -   205. Compound or construct according to any of aspects 151 to 204,     which is a multispecific construct. -   206. Compound or construct according to any of aspects 151 to 205,     which has an increased half-life, compared to the corresponding     amino acid sequence according to any of aspects 1 to 103 per se or     Nanobody according to any of aspects 104 to 150 per se,     respectively. -   207. Compound or construct according to any of aspects 151 to 206,     in which said one or more other groups, residues, moieties or     binding units provide the compound or construct with increased     half-life, compared to the corresponding amino acid sequence     according to any of aspects 1 to 103 per se or Nanobody according to     any of aspects 104 to 150 per se, respectively. -   208. Compound or construct according to aspect 207, in which said     one or more other groups, residues, moieties or binding units that     provide the compound or construct with increased half-life is chosen     from the group consisting of serum proteins or fragments thereof,     binding units that can bind to serum proteins, an Fc portion, and     small proteins or peptides that can bind to serum proteins. -   209. Compound or construct according to aspect 207, in which said     one or more other groups, residues, moieties or binding units that     provide the compound or construct with increased half-life is chosen     from the group consisting of human serum albumin or fragments     thereof. -   210. Compound or construct according to aspect 207, in which said     one or more other groups, residues, moieties or binding units that     provides the compound or construct with increased half-life are     chosen from the group consisting of binding units that can bind to     serum albumin (such as human serum albumin) or a serum     immunoglobulin (such as IgG). -   211. Compound or construct according to aspect 207, in which said     one or more other groups, residues, moieties or binding units that     provides the compound or construct with increased half-life are     chosen from the group consisting of domain antibodies, amino acid     sequences that are suitable for use as a domain antibody, single     domain antibodies, amino acid sequences that are suitable for use as     a single domain antibody, “dAb”'s, amino acid sequences that are     suitable for use as a dAb, or Nanobodies that can bind to serum     albumin (such as human serum albumin) or a serum immunoglobulin     (such as IgG). -   212. Compound or construct according to aspect 207, in which said     one or more other groups, residues, moieties or binding units that     provides the compound or construct with increased half-life is a     Nanobody that can bind to serum albumin (such as human serum     albumin) or a serum immunoglobulin (such as IgG). -   213. Compound or construct according to any of aspects 206 to 212,     that has a serum half-life that is at least 1.5 times, preferably at     least 2 times, such as at least 5 times, for example at least 10     times or more than 20 times, greater than the half-life of the     corresponding amino acid sequence according to any of aspects 1 to     103 per se or Nanobody according to any of aspects 104 to 150 per     se, respectively. -   214. Compound or construct according to any of aspects 206 to 213,     that has a serum half-life that is increased with more than 1 hours,     preferably more than 2 hours, more preferably more than 6 hours,     such as more than 12 hours, or even more than 24, 48 or 72 hours,     compared to the corresponding amino acid sequence according to any     of aspects 1 to 103 per se or Nanobody according to any of aspects     104 to 150 per se, respectively. -   215. Compound or construct according to any of aspects 206 to 214,     that has a serum half-life in human of at least about 12 hours,     preferably at least 24 hours, more preferably at least 48 hours,     even more preferably at least 72 hours or more; for example, of at     least 5 days (such as about 5 to 10 days), preferably at least 9     days (such as about 9 to 14 days), more preferably at least about 10     days (such as about 10 to 15 days), or at least about 11 days (such     as about 11 to 16 days), more preferably at least about 12 days     (such as about 12 to 18 days or more), or more than 14 days (such as     about 14 to 19 days). -   216. Compound or construct according to any of aspects 151 to 215,     in which said one or more other groups, residues, moieties or     binding units are one or more constant domains. -   217. Compound or construct according to aspect 216, wherein said one     or more constant domains are two constant domains that can be used     as part of/to form an Fc portion. -   218. Compound or construct according to any of aspects 216 or 217,     wherein said one or more constant domains are three constant domains     that can be used as part of/to form an Fc portion. -   219. Compound or construct according to any of aspects 216 to 218,     wherein said one or more constant domains confer one or more     effector functions to the compound or construct. -   220. Compound or construct according to any of aspects 216 to 219,     wherein said one or more constant domains confer the ability to bind     to one or more Fc receptors. -   221. Compound or construct according to any of aspects 216 to 220,     in which said one or more amino acid sequences or Nanobodies are     linked to the Fc portion via a linker or hinge region. -   222. Compound or construct according to any of aspects 216 to 221,     wherein said one or more constant domains are from IgG (e.g. from     IgG1, IgG2, IgG3 or IgG4). -   223. Compound or construct according to any of aspects 216 to 221,     wherein said one or more constant domains are from human Ig such as     IgA, IgD or IgM. -   224. Compound or construct according to any of aspects 216 to 221,     wherein said one or more constant domains are from IgE. -   225. Compound or construct that comprises two amino acids according     to any of aspects 1 to 103 or Nanobodies according to any of aspects     104 to 150 and an Fc portion that is capable of effecting one or     more IgE-mediated immune responses and/or that is capable of binding     to either the FcεRI receptor and/or the FcεRII receptor. -   226. Compound or construct according to aspect 225, comprising two     polypeptide chains, in which each polypeptide chain comprises at     least one amino acid sequence according to any of aspects 1 to 103     or at least one Nanobody according to any of aspects 104 to 150 that     is linked, optionally via a suitable linker or hinge region, to one     or more constant domains, in which the constant domains from the     first polypeptide chain and the second polypeptide chains together     form an Fc portion that is capable of effecting one or more     IgE-mediated immune responses and/or that is capable of binding to     either the FcεRI receptor and/or the FcεRII receptor. -   227. Compound or construct according to any of aspects 224 to 226,     in which the Fc portion is capable of binding to FcεRI with an     affinity (expressed as the K_(a) value) better than 10⁶ M⁻¹,     preferably better than 10⁸ M⁻¹, more preferably better than 10⁹ M⁻¹,     such as with a K_(a) value of about 10¹⁰ M⁻¹ or 10¹¹ M⁻¹. -   228. Compound or construct according to any of aspects 224 to 227,     in which the Fc portion is capable of binding to FcεRII with an     affinity (expressed as the K_(a) value) better than 10⁶ M⁻¹,     preferably better than 10⁷ M⁻¹, such as with a K_(a) value of about     10⁸ M⁻¹. -   229. Compound or construct according to any of aspects 224 to 228,     in which the Fc portion comprises at least one or more parts,     fragments, amino acid stretches or domains of the Fc portion of IgE. -   230. Compound or construct according to any of aspects 224 to 229,     in which the Fc portion comprises at least one or more of those     parts, fragments, amino acid stretches or domains of the Fc portion     of IgE that allow IgE to bind to its receptors. -   231. Compound or construct according to any of aspects 224 to 230,     in which the Fc portion at least comprises C_(ε)4 (or a suitable     part of fragment thereof), and optionally also comprises C_(ε)3 (or     a suitable part of fragment thereof) and/or C_(ε)2 (or a suitable     part of fragment thereof). -   232. Compound or construct according to any of aspects 224 to 231,     in which the Fc portion essentially consist of three constant     domains. -   233. Compound or construct according to any of aspects 224 to 232,     in which the parts, fragments, amino acid stretches or domains that     make up the IgE-derived Fc portion are preferably all or essentially     all derived from human Fc portions. -   234. Compound or construct according to any of aspects 224 to 233,     in which the IgE-derived Fc portion is from human IgE. -   235. Monovalent construct, comprising or essentially consisting of     one amino acid sequence according to any of aspects 1 to 103 and/or     one Nanobody according to any of aspects 104 to 150. -   236. Monovalent construct according to aspect 235, in which said     amino acid sequence is chosen from the group consisting of domain     antibodies, amino acid sequences that are suitable for use as a     domain antibody, single domain antibodies, amino acid sequences that     are suitable for use as a single domain antibody, “dAb”'s, amino     acid sequences that are suitable for use as a dAb, or Nanobodies. -   237. Monovalent construct, comprising or essentially consisting of     one Nanobody according to any of aspects 104 to 150. -   238. Monovalent construct, that is chosen from the group consisting     of SEQ ID NO's: 2051-2325 or from the group consisting of amino acid     sequences that have more than 80%, preferably more than 90%, more     preferably more than 95%, such as 99% or more sequence identity (as     defined herein) with at least one of the amino acid sequences of SEQ     ID NO's: 2051-2325. -   239. Use of a monovalent construct according to any of aspects 235     to 238, in preparing a multivalent compound or construct according     to any of aspects 151 to 234. -   240. Use of a monovalent construct according to aspect 239, in     preparing a multiparatopic construct such as a biparatopic     construct. -   241. Use of a monovalent construct according to any of aspects 239     or 240, wherein the monovalent construct is used as a binding domain     or binding unit in preparing a multivalent construct comprising two     or more binding units. -   242. Use of a monovalent construct according to any of aspects 239     to 241, in preparing a multivalent construct that exhibits     intramolecular binding compared to intermolecular binding. -   243. Use of a monovalent construct according to any of aspects 239     to 242, as a binding domain or binding unit in preparing a     multivalent construct, wherein the binding domains or binding units     are linked via a linker such that the multivalent construct     preferably exhibits intramolecular binding compared to     intermolecular binding. -   244. Use of a monovalent construct according to any of aspects 239     to 243, wherein the monovalent construct is directed against the     Omnitarg binding site on HER2 and/or is capable of competing with     Omnitarg for binding to HER-2. -   245. Use of a monovalent construct according to any of aspects 239     to 244, wherein the monovalent construct is directed against domain     II of HER2. -   246. Use of a monovalent construct according to any of aspects 239     to 245, wherein the monovalent construct is directed against the     middle of domain II of HER2. -   247. Use of a monovalent construct according to any of aspects 239     to 243, wherein the monovalent construct is directed against the     Herceptin® binding site on HER2. -   248. Use of a monovalent construct according to any of aspects 239     to 243 or 247, wherein the monovalent construct is directed against     domain IV of HER2. -   249. Use of a monovalent construct according to any of aspects 239     to 243 or 247 to 248, wherein the monovalent construct is directed     against the C-terminus of domain IV of HER2. -   250. Use of two monovalent constructs according to any of aspects     239 to 249, wherein a first monovalent construct is directed against     the Omnitarg binding site on HER2 (and in particular against domain     II of HER2, and more in particular against the middle of domain II     of HER2) and/or is capable of competing with Omnitarg for binding to     HER-2 and wherein the second monovalent construct is directed     against the Herceptin® binding site on HER2 (and in particular     against domain IV of HER2, and more in particular against the     C-terminus of domain IV of HER2) and/or is capable of competing with     Herceptin® for binding to HER-2. -   251. Nucleic acid or nucleotide sequence, that encodes an amino acid     sequence according to any of aspects 1 to 103, a Nanobody according     to any of aspects 104 to 150, a compound or construct according to     any of aspects 151 to 234 that is such that it can be obtained by     expression of a nucleic acid or nucleotide sequence encoding the     same, or a monovalent construct according to any of aspects 235 to     238. -   252. Nucleic acid or nucleotide sequence according to aspect 251,     that is in the form of a genetic construct. -   253. Use of a nucleic acid or nucleotide sequence according to     aspect 251, that encodes a monovalent construct according to any of     aspects 235 to 238, for the preparation of a genetic construct that     encodes a multivalent construct according to any of aspects 151 to     234. -   254. Use of a nucleic acid or nucleotide sequence according to     aspect 253, wherein the genetic construct encodes a multiparatopic     (such as a biparatopic) construct. -   255. Host or host cell that expresses, or that under suitable     circumstances is capable of expressing, an amino acid sequence     according to any of aspects 1 to 103, a Nanobody according to any of     aspects 104 to 150, a compound or construct according to any of     aspects 151 to 234 that is such that it can be obtained by     expression of a nucleic acid or nucleotide sequence encoding the     same, or a monovalent construct according to any of aspects 235 to     238; and/or that comprises a nucleic acid or nucleotide sequence     according to aspect 251, or a genetic construct according to aspect     252. -   256. Method for producing an amino acid sequence according to any of     aspects 1 to 103, a Nanobody according to any of aspects 104 to 150,     a compound or construct according to any of aspects 151 to 234 that     is such that it can be obtained by expression of a nucleic acid or     nucleotide sequence encoding the same, or a monovalent construct     according to any of aspects 235 to 238, said method at least     comprising the steps of:     -   a) expressing, in a suitable host cell or host organism or in         another suitable expression system, a nucleic acid or nucleotide         sequence according to aspect 251, or a genetic construct         according to aspect 252     -   optionally followed by:     -   b) isolating and/or purifying the amino acid sequence according         to any of aspects 1 to 103, the Nanobody according to any of         aspects 104 to 150, the compound or construct according to any         of aspects 151 to 234 that is such that it can be obtained by         expression of a nucleic acid or nucleotide sequence encoding the         same, or the monovalent construct according to any of aspects         235 to 238 thus obtained. -   257. Method for producing an amino acid sequence according to any of     aspects 1 to 103, a Nanobody according to any of aspects 104 to 150,     a compound or construct according to any of aspects 151 to 234 that     is such that it can be obtained by expression of a nucleic acid or     nucleotide sequence encoding the same, or a monovalent construct     according to any of aspects 235 to 238, said method at least     comprising the steps of:     -   a) cultivating and/or maintaining a host or host cell according         to aspect 255 under conditions that are such that said host or         host cell expresses and/or produces at least one amino acid         sequence according to any of aspects 1 to 103, Nanobody         according to any of aspects 104 to 150, compound or construct         according to any of aspects 151 to 234 that is such that it can         be obtained by expression of a nucleic acid or nucleotide         sequence encoding the same, or a monovalent construct according         to any of aspects 235 to 238,     -   optionally followed by:     -   b) isolating and/or purifying the amino acid sequence according         to any of aspects 1 to 103, the Nanobody according to any of         aspects 104 to 150, the compound or construct according to any         of aspects 151 to 234 that is such that it can be obtained by         expression of a nucleic acid or nucleotide sequence encoding the         same, or the monovalent construct according to any of aspects         235 to 238 thus obtained. -   258. Method for preparing and/or generating a multiparatopic (such     as e.g. biparatopic, triparatopic, etc.) construct according to any     of aspects 161 to 204, said method comprising at least the steps of:     -   a) providing a nucleic acid sequence according to aspect 251,         encoding a first HER2 binding amino acid sequence, fused to a         set, collection or library of nucleic acid sequences encoding         amino acid sequences;     -   b) screening said set, collection or library of nucleic acid         sequences for nucleic acid sequences that encode a second amino         acid sequence that can bind to and/or has affinity for an         antigenic determinant on HER2 different from the antigenic         determinant recognized by the first HER2 binding amino acid         sequence;     -   and     -   c) isolating the nucleic acid sequence encoding an HER2 binding         amino acid sequence fused to the nucleic acid sequence obtained         in b), followed by expressing the encoded construct. -   259. Method for preparing and/or generating a biparatopic construct     according to any of aspects 161 to 204, said method comprising at     least the steps of:     -   a) providing a set, collection or library of nucleic acid         sequences, in which each nucleic acid sequence in said set,         collection or library encodes a fusion protein that comprises a         first amino acid sequence that can bind to and/or has affinity         for a first antigenic determinant, part, domain or epitope on         HER2 that is fused (optionally via a linker sequence) to a         second amino acid sequence, in which essentially each second         amino acid sequence (or most of these) is a different member of         a set, collection or library of different amino acid sequences;     -   b) screening said set, collection or library of nucleic acid         sequences for nucleic acid sequences that encode an amino acid         sequence that can bind to and/or has affinity for a second         antigenic determinant, part, domain or epitope on HER2 different         from the first antigenic determinant, part, domain or epitope on         HER-2;     -   and     -   c) isolating the nucleic acid sequences that encode an amino         acid sequence that can bind to and/or has affinity for a second         antigenic determinant, part, domain or epitope on HER2 different         from the first antigenic determinant, part, domain or epitope on         HER-2, obtained in b), optionally followed by expressing the         encoded amino acid sequence. -   260. Method according to aspect 259, wherein the first amino acid is     also encoded by a set, collection or library of nucleic acid     sequences and wherein, in step b), said set, collection or library     of nucleic acid sequences is screened for nucleic acid sequences     that encode an amino acid sequence that can bind to and/or has     affinity for the first antigenic determinant, part, domain or     epitope on HER2. -   261. Method according to aspect 260, wherein the screening in     step b) is performed in a single step. -   262. Method according to aspect 260, wherein the screening in     step b) is performed in subsequent steps. -   263. Method according to any of aspects 258 to 262, wherein the     first amino acid sequence used in step a) is preferably such     that (i) it can bind to and/or has affinity for the Herceptin®     binding site on HER2 (and in particular domain IV of HER2, more in     particular the C-terminus of domain IV of HER2) and/or (ii) competes     with Herceptin® for binding to HER-2. -   264. Method according to any of aspects 258 to 263, wherein in step     b), the set, collection or library of nucleic acid sequences is     screened for nucleic acid sequences that encode (i) an amino acid     sequence that can bind to and/or has affinity for the Omnitarg     binding site on HER2 (and in particular domain TI of HER2, more in     particular the middle of domain II of HER2) and/or (ii) an amino     acid sequence that can compete with Omnitarg or Omnitarg Fab for     binding to HER-2. -   265. Method according to any of aspects 258 to 264, wherein the     first amino acid sequence used in step a) is preferably such     that (i) it can bind to and/or has affinity for the Omnitarg binding     site on HER2 (and in particular domain II of HER2, more in     particular the middle of domain H of HER2) and/or (ii) competes with     Omnitarg for binding to HER-2. -   266. Method according to any of aspects 258 to 265, wherein in step     b), the set, collection or library of nucleic acid sequences is     screened for nucleic acid sequences that encode (i) an amino acid     sequence that can bind to and/or has affinity for the Herceptin®     binding site on HER2 (and in particular domain IV of HER2, more in     particular the C-terminus of domain IV of HER2) and/or (ii) an amino     acid sequence that can compete with Herceptin® for binding to HER-2. -   267. Method according to any of aspects 258 to 266, wherein in step     b), the set, collection or library of nucleic acid sequences is     screened for nucleic acid sequences that both (i) encode an amino     acid sequence that can bind to and/or has affinity for the Omnitarg     binding site on HER2 (and in particular domain II of HER2, more in     particular the middle of domain II of HER2) and/or that can compete     with Omnitarg or Omnitarg Fab for binding to HER-2 and that     also (ii) encode an amino acid sequence that can bind to and/or has     affinity for the Herceptin® binding site on HER2 (and in particular     domain IV of HER2, more in particular the C-terminus of domain IV of     HER2) and/or that can compete with Herceptin® for binding to HER-2. -   268. Method according to aspect 267, wherein the screening in     step b) is performed in a single step. -   269. Method according to aspect 267, wherein the screening in     step b) is performed in a subsequent steps. -   270. Method according to any of aspects 258 to 269, wherein the     screening in step b) is performed in the presence of Herceptin®     and/or Omnitarg. -   271. Method for screen for suitable and/or optimal linker lengths     for linking a first and a second amino acid sequence in a     biparatopic constructs according to any of aspects 161 to 204,     wherein said method comprises at least the steps of:     -   d) providing a set, collection or library of nucleic acid         sequences, in which each nucleic acid sequence in said set,         collection or library encodes a fusion protein that comprises a         first amino acid sequence that can bind to and/or has affinity         for a first antigenic determinant, part, domain or epitope on         HER2 that is fused via a linker sequence to a second amino acid         sequence that has can bind to and/or has affinity for a second         antigenic determinant, part, domain or epitope on HER2 (which         may be the same or different as the first antigenic determinant,         part, domain or epitope on HER2), in which essentially each         nucleic acid sequence (or most of these) encodes a fusion         protein with a different linker sequence so as to provide a set,         collection or library encoding different fusion proteins;     -   e) screening said set, collection or library of nucleic acid         sequences for nucleic acid sequences that encode an amino acid         sequence that can bind to and/or has affinity for the first and         second antigenic determinant, part, domain or epitope on HER2;     -   and     -   f) isolating the nucleic acid sequences that encode an amino         acid sequence that can bind to and/or has affinity for the first         and second antigenic determinant, part, domain or epitope on         HER2, optionally followed by expressing the encoded amino acid         sequence. -   272. Method according to aspect 271, wherein the first amino acid     sequence is an amino acid sequence that can bind to and/or has     affinity for the Omnitarg binding site on HER2 (and may in     particular domain II of HER2, more in particular the middle of     domain II of HER2) and/or that can compete with Omnitarg or Omnitarg     Fab. -   273. Method according to aspect 271, wherein the second amino acid     sequence is an amino acid sequence that can bind to and/or has     affinity for the Herceptin® binding site on HER2 (and in particular     domain IV of HER2, more in particular the C-terminus of domain IV of     HER2) and/or that can compete with Herceptin® for binding to HER-2. -   274. Method according to aspect 271, wherein the first amino acid     sequence is an amino acid sequence that can bind to and/or has     affinity for the Omnitarg binding site on HER2 (and may in     particular domain. II of HER2, more in particular the middle of     domain II of HER2) and/or that can compete with Omnitarg or Omnitarg     Fab and wherein the second amino acid sequence is an amino acid     sequence that can bind to and/or has affinity for the Herceptin®     binding site on HER2 (and in particular domain IV of HER2, more in     particular the C-terminus of domain IV of HER2) and/or that can     compete with Herceptin® for binding to HER-2. -   275. Method according to any of aspects 271 to 274, wherein the     screening in step b) is performed in a single step. -   276. Method according to any of aspects 271 to 274, wherein the     screening in step b) is performed in subsequent steps. -   277. Method according to any of aspects 271 to 276, wherein the     screening in step b) is performed in the presence of Herceptin®     and/or Omnitarg. -   278. Method for preparing and/or generating biparatopic constructs     according to any of aspects 161 to 204, said method comprising at     least the steps of:     -   a) providing a set, collection or library of nucleic acid         sequences encoding amino acid sequences;     -   b) screening said set, collection or library of nucleic acid         sequences for a set, collection or library of nucleic acid         sequences that encode an amino acid sequence that can bind to         and/or has affinity for HER2;     -   c) ligating said set, collection or library of nucleic acid         sequences that encode an amino acid sequence that can bind to         and/or has affinity for HER2 to another nucleic acid sequence         that encodes an amino acid sequence that can bind to and/or has         affinity for HER2 (e.g. a nucleic acid sequence that encodes an         amino acid sequence that competes with Herceptin® for binding         HER2);     -   and     -   d) from the set, collection or library of nucleic acid sequences         obtained in c), isolating the nucleic acid sequences encoding a         biparatopic amino acid sequence that can bind to and/or has         affinity for HER2 (and e.g. further selecting for nucleic acid         sequences that encode a biparatopic amino acid sequence that         antagonizes with higher potency compared to the monovalent amino         acid sequences), followed by expressing the encoded amino acid         sequence. -   279. Method for preparing and/or generating biparatopic constructs     according to any of aspects, said method comprising at least the     steps of:     -   a) providing a first set, collection or library of nucleic acid         sequences encoding amino acid sequences;     -   b) screening said first set, collection or library of nucleic         acid sequences for a nucleic acid sequence that encodes an amino         acid sequence that can bind to and/or has affinity for a first         antigenic determinant, part, domain or epitope on HER2;     -   c) ligating the nucleic acid sequence encoding said amino acid         sequence that can bind to and/or has affinity for a first         antigenic determinant, part, domain or epitope on HER2 obtained         in b) to another set, collection or library of nucleic acid         sequences encoding amino acid sequences to obtain a set,         collection or library of nucleic acid sequences that encode         fusion proteins;     -   d) screening said set, collection or library of nucleic acid         sequences obtained in step c) for a nucleic acid sequence that         encodes an amino acid sequence that can bind to and has affinity         for a second antigenic determinant, part, domain or epitope on         HER2 which is the same or different from the first antigenic         determinant, part, domain or epitope on HER-2;     -   and     -   e) isolating the nucleic acid sequence that encodes an amino         acid sequence that can bind to and/or has affinity for the first         and second antigenic determinant, part, domain or epitope on         HER2, optionally followed by expressing the encoded amino acid         sequence. -   280. Method according to aspect 279, wherein in step b), the set,     collection or library of nucleic acid sequences is screened for     nucleic acid sequences that encode a first amino acid sequence     that (i) can bind to and/or has affinity for the Herceptin® binding     site on HER2 (and in particular domain IV of HER2, more in     particular the C-terminus of domain IV of HER2) and/or (ii) competes     with Herceptin® for binding to HER-2. -   281. Method according to aspect 279, wherein in step d), the set,     collection or library of nucleic acid sequences is screened for     nucleic acid sequences that encode a second amino acid sequence     that (i) can bind to and/or has affinity for the Omnitarg binding     site on HER2 (and in particular domain. II of HER2, more in     particular the middle of domain II of HER2) and/or (ii) that can     compete with Omnitarg or Omnitarg Fab for binding to HER-2. -   282. Method according to aspect 279, wherein in step b), the set,     collection or library of nucleic acid sequences is screened nucleic     acid sequences that encode a first amino acid sequence that (i) can     bind to and/or has affinity for the Herceptin® binding site on HER2     (and in particular domain IV of HER2, more in particular the     C-terminus of domain IV of HER2) and/or (ii) competes with     Herceptin® for binding to HER-2 and wherein in step d), the set,     collection or library of nucleic acid sequences is screened for     nucleic acid sequences that encode a second amino acid sequence     that (i) can bind to and/or has affinity for the Omnitarg binding     site on HER2 (and in particular domain II of HER2, more in     particular the middle of domain II of HER2) and/or (ii) that can     compete with Omnitarg or Omnitarg Fab for binding to HER-2 (or visa     versa). -   283. Method according to aspect 279, wherein in step b), the set,     collection or library of nucleic acid sequences is screened nucleic     acid sequences that encode a first amino acid sequence that (i) can     bind to and/or has affinity for the Omnitarg binding site on HER2     (and in particular domain II of HER2, more in particular the middle     of domain II of HER2) and/or (ii) competes with Omnitarg for binding     to HER-2. -   284. Method according to aspect 279, wherein in step d), the set,     collection or library of nucleic acid sequences is screened for     nucleic acid sequences that encode a second amino acid sequence     that (i) can bind to and/or has affinity for the Herceptin® binding     site on HER2 (and in particular domain IV of HER2, more in     particular the C-terminus of domain IV of HER2) and/or (ii) that can     compete with Herceptin® for binding to HER-2. -   285. Method according to aspect 279, wherein in step b), the set,     collection or library of nucleic acid sequences is screened nucleic     acid sequences that encode a first amino acid sequence that (i) can     bind to and/or has affinity for the Omnitarg binding site on HER2     (and in particular domain H of HER2, more in particular the middle     of domain II of HER2) and/or (ii) competes with Omnitarg for binding     to HER-2 and wherein in step d), the set, collection or library of     nucleic acid sequences is screened for nucleic acid sequences that     encode a second amino acid sequence that (i) can bind to and/or has     affinity for the Herceptin® binding site on HER2 (and in particular     domain IV of HER2, more in particular the C-terminus of domain. IV     of HER2) and/or (ii) that can compete with Herceptin® for binding to     HER-2. -   286. Method according to aspect 285, wherein the screening in     steps b) and/or d) is performed in the presence of Herceptin® and/or     Omnitarg. -   287. Method for preparing and/or generating a biparatopic constructs     according to any of aspects 161 to 204, said method comprising at     least the steps of linking two or more monovalent amino acid     sequences or monovalent construct according to any of aspects 235 to     238 and for example one or more linkers. -   288. Method according to aspect 287, comprising the steps of:     -   a) linking two or more nucleic acid sequences according to         aspect 251, encoding a monovalent construct according to any of         aspects 235 to 238 (and also for example nucleic acids encoding         one or more linkers and further one or more further elements of         genetic constructs known per se) to obtain a genetic construct         according to aspect 252;     -   b) expressing, in a suitable host cell or host organism or in         another suitable expression system, the genetic construct         obtained in a)     -   optionally followed by:     -   c) isolating and/or purifying the biparatopic constructs         according to any of aspects 161 to 204 thus obtained. -   289. Composition, comprising at least one amino acid sequence     according to any of aspects 1 to 103, Nanobody according to any of     aspects 104 to 150, compound or construct according to any of     aspects 151 to 234, monovalent construct according to any of aspects     235 to 238, or nucleic acid or nucleotide sequence according to     aspects 251 to 252. -   290. Composition according to aspect 289, which is a pharmaceutical     composition. -   291. Composition according to aspect 289, which is a pharmaceutical     composition, that further comprises at least one pharmaceutically     acceptable carrier, diluent or excipient and/or adjuvant, and that     optionally comprises one or more further pharmaceutically active     polypeptides and/or compounds. -   292. Method for the prevention and/or treatment of at least one     cancer and/or tumor, said method comprising administering, to a     subject in need thereof, a pharmaceutically active amount of at     least one amino acid sequence according to any of aspects 1 to 103,     Nanobody according to any of aspects 104 to 150, compound or     construct according to any of aspects 151 to 234, monovalent     construct according to any of aspects 235 to 238, or composition     according to aspect 290 or 291. -   293. Method for the prevention and/or treatment of at least one     disease or disorder that is associated with HER2, with its     biological or pharmacological activity, and/or with the biological     pathways or signalling in which HER2 is involved, said method     comprising administering, to a subject in need thereof, a     pharmaceutically active amount of at least one amino acid sequence     according to any of aspects 1 to 103, Nanobody according to any of     aspects 104 to 150, compound or construct according to any of     aspects 151 to 234, monovalent construct according to any of aspects     235 to 238, or composition according to aspect 290 or 291. -   294. Method for the prevention and/or treatment of at least one     disease or disorder that can be prevented and/or treated by     administering, to a subject in need thereof, an amino acid sequence     according to any of aspects 1 to 103, a Nanobody according to any of     aspects 104 to 150, a compound or construct according to any of     aspects 151 to 234, or a monovalent construct according to any of     aspects 235 to 238, said method comprising administering, to a     subject in need thereof, a pharmaceutically active amount of at     least one amino acid sequence according to any of aspects 1 to 103,     Nanobody according to any of aspects 104 to 150, compound or     construct according to any of aspects 151 to 234, monovalent     construct according to any of aspects 235 to 238, or composition     according to aspect 290 or 291. -   295. Method for immunotherapy, said method comprising administering,     to a subject in need thereof, a pharmaceutically active amount of at     least one amino acid sequence according to any of aspects 1 to 103,     Nanobody according to any of aspects 104 to 150, compound or     construct according to any of aspects 151 to 234, monovalent     construct according to any of aspects 235 to 238, or composition     according to aspect 290 or 291. -   296. Use of an amino acid sequence according to any of aspects 1 to     103, a Nanobody according to any of aspects 104 to 150, a compound     or construct according to any of aspects 151 to 234, or a monovalent     construct according to any of aspects 235 to 238 in the preparation     of a pharmaceutical composition for prevention and/or treatment of     at least one cancer and/or tumor; and/or for use in one or more of     the methods according to aspects 292 to 295. -   297. An amino acid sequence according to any of aspects 1 to 103, a     Nanobody according to any of aspects 104 to 150, a compound or     construct according to any of aspects 151 to 234, or a monovalent     construct according to any of aspects 235 to 238 for use in the     prevention and/or treatment of at least one cancer and/or tumor;     and/or for use in one or more of the methods according to aspects     292 to 295. -   298. Part or fragment of an amino acid sequence according to any of     aspects 1 to 103, or of a Nanobody according to any of aspects 104     to 150. -   299. Part or fragment according to aspect 298, that can specifically     bind to HER2. -   300. Part or fragment according to aspect 299, wherein said part or     fragment competes with Herceptin® for binding to HER2. -   301. Part of fragment according to any of aspects 299 or 300,     wherein said part or fragment inhibits and/or blocks binding of     Herceptin® to HER2. -   302. Part or fragment according to any of aspects 299 to 301,     wherein said part or fragment is directed against the Herceptin®     binding site on HER2. -   303. Part of fragment according to any of aspects 299 to 302,     wherein said part or fragment specifically binds to domain IV of     HER2. -   304. Part of fragment according to aspect 299, wherein said part or     fragment competes with Omnitarg for binding to HER2. -   305. Part of fragment according to any of aspects 299 or 304,     wherein said part or fragment inhibits and/or blocks binding of     Omnitarg to HER2. -   306. Part of fragment according to any of aspects 304 or 305,     wherein said part or fragment is directed against the Omnitarg     binding site on HER2. -   307. Part of fragment according to any of aspects 304 to 306,     wherein said part or fragment specifically binds to domain II of     HER2. -   308. Part of fragment according to any of aspects 299 to 307,     wherein said part or fragment competes with Herceptin® and Omnitarg     for binding to HER2. -   309. Part of fragment according to any of aspects 299 to 308,     wherein said part or fragment inhibits and/or blocks binding of     Herceptin® and Omnitarg to HER2. -   310. Part of fragment according to any of aspects 299 to 309,     wherein said part or fragment inhibits and/or blocks tumor cell     proliferation. -   311. Part of fragment according to any of aspects 299 to 310,     wherein said part or fragment inhibits, downregulates and/or blocks     cell signalling. -   312. Part of fragment according to any of aspects 299 to 311,     wherein said part or fragment induces apoptosis in tumor cells. -   313. Part of fragment according to any of aspects 299 to 312,     wherein said part or fragment inhibits and/or blocks     heterodimerization between ERBB receptors. -   314. Part of fragment according to any of aspects 299 to 313,     wherein said part or fragment inhibits and/or blocks ligand     activation of an ERbB hetero-oligomer comprising HER2 and HER3, HER4     or EGFR. -   315. Part of fragment according to any of aspects 299 to 314,     wherein said part or fragment inhibits and/or blocks tumor     vascularisation. -   316. Part of fragment according to any of aspects 299 to 315,     wherein said part or fragment recruits immune effector cells such as     macrophages and monocytes to the tumor. -   317. Part of fragment according to any of aspects 299 to 316,     wherein said part or fragment inhibits and/or blocks TNF induced     signalling and/or cell proliferation. -   318. Part of fragment according to any of aspects 299 to 317,     wherein said part or fragment downregulates HER2 levels and/or     downregulates HER2-mediated signalling pathways. -   319. Part of fragment according to any of aspects 299 to 318,     wherein said part or fragment inhibits and/or blocks     metalloproteinase-mediated HER2 ectodomain shedding. -   320. Part of fragment according to any of aspects 299 to 319,     wherein said part or fragment inhibits, downregulates and/or blocks     ligand-mediated ErbB signalling. -   321. Part of fragment according to any of aspects 299 to 320,     wherein said part or fragment inhibits and/or blocks HER2 ectodomain     cleavage. -   322. Part of fragment according to any of aspects 299 to 321,     wherein said part or fragment inhibits and/or blocks     Heregulin-mediated activation of MAPK/Erk1/2. -   323. Part of fragment according to any of aspects 299 to 322,     wherein said part or fragment inhibits and/or blocks PI3K/Akt     signalling. -   324. Part of fragment according to any of aspects 299 to 323,     wherein said part or fragment modulates HER2 or HER2 mediated     signalling via the same mechanism of action as Herceptin®. -   325. Part of fragment according to any of aspects 299 to 324,     wherein said part or fragment modulates HER2 or HER2 mediated     signalling via the same mechanism of action as Omnitarg. -   326. Part of fragment according to any of aspects 299 to 325, that     can specifically bind to HER2 with a dissociation constant (K_(D))     of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²     moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre. -   327. Part or fragment according to any of aspects 299 to 326, that     can specifically bind to HER2 with a rate of association     (k_(on)-rate) of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,     preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably     between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and     10⁷ M⁻¹ s⁻¹. -   328. Part or fragment according to any of aspects 299 to 327, that     can specifically bind to HER2 with a rate of dissociation (k_(off)     rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and     10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as     between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹. -   329. Compound or construct, that comprises or essentially consists     of one or more parts or fragments according to any of aspects 299 to     328, and optionally further comprises one or more other groups,     residues, moieties or binding units, optionally linked via one or     more linkers. -   330. Compound or construct according to aspect 329, in which said     one or more other groups, residues, moieties or binding units are     amino acid sequences. -   331. Compound or construct according to aspect 329, in which said     one or more linkers, if present, are one or more amino acid     sequences. -   332. Nucleic acid or nucleotide sequence, that encodes a part or     fragment according to any of aspects 299 to 328 or a compound or     construct according to any of aspects 329 to 331 that is such that     it can be obtained by expression of a nucleic acid or nucleotide     sequence encoding the same. -   333. Composition, comprising at least one part or fragment according     to any of aspects 299 to 328, compound or construct according to any     of aspects 329 to 331, or nucleic acid or nucleotide sequence     according to aspect 332. -   334. Derivative of an amino acid sequence according to any of     aspects 1 to 103, or of a Nanobody according to any of aspects 104     to 150. -   335. Derivative according to aspect 334, that can specifically bind     to HER2. -   336. Derivative according to aspect 335, wherein said derivative     competes with Herceptin® for binding to HER2. -   337. Derivative according to any of aspects 335 or 336, wherein said     derivative inhibits and/or blocks binding of Herceptin® to HER2. -   338. Derivative according to any of aspects 335 to 337, wherein said     derivative is directed against the Herceptin® binding site on HER2. -   339. Derivative according to any of aspects 335 to 338, wherein said     derivative specifically binds to domain IV of HER2. -   340. Derivative according to aspect 335, wherein said derivative     competes with Omnitarg for binding to HER2. -   341. Derivative according to any of aspects 335 or 340, wherein said     derivative inhibits and/or blocks binding of Omnitarg to HER2. -   342. Derivative according to any of aspects 340 or 341, wherein said     derivative is directed against the Omnitarg binding site on HER2. -   343. Derivative according to any of aspects 340 to 342, wherein said     derivative specifically binds to domain II of HER2. -   344. Derivative according to any of aspects 335 to 343, wherein said     derivative competes with Herceptin® and Omnitarg for binding to     HER2. -   345. Derivative according to any of aspects 335 to 344, wherein said     derivative inhibits and/or blocks binding of Herceptin® and Omnitarg     to HER2. -   346. Derivative according to any of aspects 335 to 345, wherein said     derivative inhibits and/or blocks tumor cell proliferation. -   347. Derivative according to any of aspects 335 to 346, wherein said     derivative inhibits, downregulates and/or blocks cell signalling. -   348. Derivative according to any of aspects 335 to 347, wherein said     derivative induces apoptosis in tumor cells. -   349. Derivative according to any of aspects 335 to 348, wherein said     derivative inhibits and/or blocks heterodimerization between ERBB     receptors. -   350. Derivative according to any of aspects 335 to 349, wherein said     derivative inhibits and/or blocks ligand activation of an ERbB     hetero-oligomer comprising HER2 and HER3, HER4 or EGFR. -   351. Derivative according to any of aspects 335 to 350, wherein said     derivative inhibits and/or blocks tumor vascularisation. -   352. Derivative according to any of aspects 335 to 351, wherein said     derivative recruits immune effector cells such as macrophages and     monocytes to the tumor. -   353. Derivative according to any of aspects 335 to 352, wherein said     derivative inhibits and/or blocks TNF induced signalling and/or cell     proliferation. -   354. Derivative according to any of aspects 335 to 353, wherein said     derivative down-regulates HER2 levels and/or downregulates     HER2-mediated signalling pathways. -   355. Derivative according to any of aspects 335 to 354, wherein said     derivative inhibits and/or blocks metalloproteinase-mediated HER2     ectodomain shedding. -   356. Derivative according to any of aspects 335 to 355, wherein said     derivative inhibits, downregulates and/or blocks ligand-mediated     ErbB signalling. -   357. Derivative according to any of aspects 335 to 356, wherein said     derivative inhibits and/or blocks HER2 ectodomain cleavage. -   358. Derivative according to any of aspects 335 to 357, wherein said     derivative inhibits and/or blocks Heregulin-mediated activation of     MAPK/Erk1/2. -   359. Derivative according to any of aspects 335 to 358, wherein said     derivative inhibits and/or blocks PI3K/Akt -   360. Derivative according to any of aspects 335 to 359, wherein said     derivative modulates HER2 or HER2 mediated signalling via the same     mechanism of action as Herceptin®. -   361. Derivative according to any of aspects 335 to 360, wherein said     derivative modulates HER2 or HER2 mediated signalling via the same     mechanism of action as Omnitarg. -   362. Derivative according to any of aspects 335 to 361, that can     specifically bind to HER2 with a dissociation constant (K_(D)) of     10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²     moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre. -   363. Derivative according to any of aspects 335 to 362, that can     specifically bind to HER2 with a rate of association (k_(on)-rate)     of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹, preferably between 10³     M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷     M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹. -   364. Derivative according to any of aspects 335 to 363, that can     specifically bind to HER2 with a rate of dissociation (k_(off) rate)     between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,     more preferably between 10⁻³ s⁻¹ and 10⁻⁶ such as between 10⁻⁴ s⁻¹     and 10⁻⁶ s⁻¹. -   365. Derivative of a compound or construct according to any of     aspects 151 to 234. -   366. Derivative according to aspect 365, that can specifically bind     to HER2. -   367. Derivative according to aspect 366, wherein said derivative     competes with Herceptin® for binding to HER2. -   368. Derivative according to any of aspects 366 or 367, wherein said     derivative inhibits and/or blocks binding of Herceptin® to HER2. -   369. Derivative according to any of aspects 366 to 368, wherein said     derivative is directed against the Herceptin® binding site on HER2. -   370. Derivative according to any of aspects 366 to 369, wherein said     derivative specifically binds to domain IV of HER2. -   371. Derivative according to aspect 366, wherein said derivative     competes with Omnitarg for binding to HER2. -   372. Derivative according to any of aspects 366 or 371, wherein said     derivative inhibits and/or blocks binding of Omnitarg to HER2. -   373. Derivative according to any of aspects 371 or 372, wherein said     derivative is directed against the Omnitarg binding site on HER2. -   374. Derivative according to any of aspects 371 to 373, wherein said     derivative specifically binds to domain II of HER2. -   375. Derivative according to any of aspects 366 to 374, wherein said     derivative competes with Herceptin® and Omnitarg for binding to     HER2. -   376. Derivative according to any of aspects 366 to 375, wherein said     derivative inhibits and/or blocks binding of Herceptin® and Omnitarg     to HER2. -   377. Derivative according to any of aspects 366 to 376, wherein said     derivative inhibits and/or blocks tumor cell proliferation. -   378. Derivative according to any of aspects 366 to 377, wherein said     derivative inhibits, downregulates and/or blocks cell signalling. -   379. Derivative according to any of aspects 366 to 378, wherein said     derivative induces apoptosis in tumor cells. -   380. Derivative according to any of aspects 366 to 379, wherein said     derivative inhibits and/or blocks heterodimerization between ERBB     receptors. -   381. Derivative according to any of aspects 366 to 380, wherein said     derivative inhibits and/or blocks ligand activation of an ERbB     hetero-oligomer comprising HER2 and HER3, HER4 or EGFR. -   382. Derivative according to any of aspects 366 to 381, wherein said     derivative inhibits and/or blocks tumor vascularisation. -   383. Derivative according to any of aspects 366 to 382, wherein said     derivative recruits immune effector cells such as macrophages and     monocytes to the tumor. -   384. Derivative according to any of aspects 366 to 383, wherein said     derivative inhibits and/or blocks TNF induced signalling and/or cell     proliferation. -   385. Derivative according to any of aspects 366 to 384, wherein said     derivative down-regulates HER2 levels and/or downregulates     HER2-mediated signalling pathways. -   386. Derivative according to any of aspects 366 to 385, wherein said     derivative inhibits and/or blocks metalloproteinase-mediated HER2     ectodomain shedding. -   387. Derivative according to any of aspects 366 to 386, wherein said     derivative inhibits, downregulates and/or blocks ligand-mediated     ErbB signalling. -   388. Derivative according to any of aspects 366 to 387, wherein said     derivative inhibits and/or blocks HER2 ectodomain cleavage. -   389. Derivative according to any of aspects 366 to 388, wherein said     derivative inhibits and/or blocks Heregulin-mediated activation of     MAPK/Erk1/2. -   390. Derivative according to any of aspects 366 to 389, wherein said     derivative inhibits and/or blocks PI3K/Akt signalling. -   391. Derivative according to any of aspects 366 to 390, wherein said     derivative modulates HER2 or HER2 mediated signalling via the same     mechanism of action as Herceptin®. -   392. Derivative according to any of aspects 366 to 391, wherein said     derivative modulates HER2 or HER2 mediated signalling via the same     mechanism of action as Omnitarg. -   393. Derivative according to any of aspects 366 to 392, that can     specifically bind to HER2 with a dissociation constant (K_(D)) of     10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²     moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter. -   394. Derivative according to any of aspects 366 to 393, that can     specifically bind to HER2 with a rate of association (k_(on)-rate)     of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹, preferably between 10³     M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹ s⁻¹ and     10⁷M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹. -   395. Derivative according to any of aspects 366 to 394, that can     specifically bind to HER2 with a rate of dissociation (k_(off) rate)     between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,     more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴     s⁻¹ and 10⁻⁶ s⁻¹. -   396. Derivative according to any of aspects 334 to 395, that has a     serum half-life that is at least 1.5 times, preferably at least 2     times, such as at least 5 times, for example at least 10 times or     more than 20 times, greater than the half-life of the corresponding     amino acid sequence according to any of aspects 1 to 103 per se,     Nanobody according to any of aspects 104 to 150 per se, or compound     or construct according to any of aspects 151 to 234 per se. -   397. Derivative according to any of aspects 334 to 396, that has a     serum half-life that is increased with more than 1 hours, preferably     more than 2 hours, more preferably more than 6 hours, such as more     than 12 hours, or even more than 24, 48 or 72 hours, compared to the     corresponding amino acid sequence according to any of aspects 1 to     103 per se or Nanobody according to any of aspects 104 to 150 per     se, respectively. -   398. Derivative according to any of aspects 334 to 397, that has a     serum half-life in human of at least about 12 hours, preferably at     least 24 hours, more preferably at least 48 hours, even more     preferably at least 72 hours or more; for example, at least 5 days     (such as about 5 to 10 days), preferably at least 9 days (such as     about 9 to 14 days), more preferably at least about 10 days (such as     about 10 to 15 days), or at least about 11 days (such as about 11 to     16 days), more preferably at least about 12 days (such as about 12     to 18 days or more), or more than 14 days (such as about 14 to 19     days). -   399. Derivative according to any of aspects 334 to 398, that is a     pegylated derivative. -   400. Compound or construct, that comprises or essentially consists     of one or more derivatives according to any of aspects 334 to 399,     and optionally further comprises one or more other groups, residues,     moieties or binding units, optionally linked via one or more     linkers. -   401. Compound or construct according to aspect 400, in which said     one or more other groups, residues, moieties or binding units are     amino acid sequences. -   402. Compound or construct according to aspect 400, in which said     one or more linkers, if present, are one or more amino acid     sequences. -   403. Nucleic acid encoding a compound or construct according to any     of aspects 400 to 402 that is such that it can be obtained by     expression of a nucleic acid or nucleotide sequence encoding the     same. -   404. Composition, comprising at least one derivative to any of     aspects 334 to 399, compound or construct according to any of     aspects 400 to 402, or nucleic acid or nucleotide sequence according     to aspect 403.

FIGURE LEGENDS

FIG. 1: Anti-HER2 humoral immune response induced after immunisation of Llama glama with HER2-overexpressing SKBR3 cells. The reactivity of pre-immune (day 0) and immune sera (day 42 and day of PBL1 take) of animals 121 and 122 immunized with whole cells was determined by ELISA using rhErbB2-Fc as antigen (see Example 3.2). A: total IgG response; B: IgG1 isotype response; C: IgG2 isotype response; D: IgG3 isotype response.

FIG. 2: HER2-specific ELISA analysis of periplasmic preparations containing myc-tagged Nanobody protein fragments from selected clones. Periplasmic preparations of soluble Nanobody protein fragments were added to wells of an ELISA plate, which had been coated with rhErbB2/Fc antigen and had been additionally blocked with PBS+1% casein. Detection was performed by a monoclonal anti-myc antibody followed by an alkaline phosphatase-conjugated polyclonal goat anti-mouse antibody. The ELISA was developed by a PNPP-substrate as described in Example 6. The OD-values (Y-axis) were measured at 405 nm by an ELISA-reader. Each bar represents an individual periplasmic extract.

FIG. 3: Flow cytometric analysis of selected clones (2A1, 2A4, 2C3, 2C5, 2D3 and 2G4). Nanobody-containing periplasmic extracts were added to ErbB2 overexpressing SKBR3 cells. Detection was performed by a monoclonal anti-myc antibody followed by a PE-labeled polyclonal anti-mouse antibody. Nanobodies binding to cells was measured by an increase in fluorescence intensity as compared to cells that were incubated with FACS buffer (PBS+10% FBS) followed by monoclonal anti-myc antibody and PE-labeled polyclonal anti-mouse antibody. Fluorescence intensity is blotted on the X-axis, the number of events on the Y-axis.

FIG. 4: Herceptin® competitive ELISA. An ELISA plate was coated with SKBR3 vesicles (5 μg/ml) and additionally blocked with PBS+1% casein. 2 nM Herceptin® was added to the wells, after which periplasmic preparations of soluble Nanobody protein fragments were added. Detection of Herceptin® binding to SKBR3 vesicles was performed by an alkaline phosphatase-conjugated AffiniPure Goat Anti-Human IgG, Fc Fragment Specific (Jackson ImmunoResearch Labs, Suffolk, UK). The ELISA was developed by a PNPP-substrate as described in Example 6. The OD-values (Y-axis) were measured at 405 nm by an ELISA-reader. Each bar represents an individual periplasmic extract. The OD value corresponding to the maximal signal represents the OD value measured for binding of Herceptin® without addition of periplasmic extract containing HER-binding Nanobody. The minimal signal represents the background staining of non-coated wells incubated only with alkaline phosphatase-conjugated AffiniPure Goat Anti-Human IgG, followed by detection using a PNPP-substrate. Controls 1-5 represent individual periplasmic extracts containing non-HER2 binding Nanobodies.

FIG. 5: Herceptin®-competitive FMAT. Dilutions of periplasmic extracts containing HER2 binding Nanobodies were tested for their ability to block the binding of Herceptin® to HER2-overexpressing SKBR3 cells as described in Example 8. (−) represents the signal obtained for binding of Alexa647-labeled Herceptin® without addition of periplasmic extract. Addition of periplasmic extract containing a non-HER2 binding Nanobody (irr) had no influence on the binding of Herceptin® to SKBR3 cells. Periplasmic extracts 2A4, 2A5, 2A6, 2B1, 2B2, 2B4, 2B5, 2C1, 2C3, 2D2 and 2D3 blocked binding of Herceptin® to HER2 with more than 80%.

FIG. 6: Herceptin®-competitive FMAT analysis. Nanobodies compete with binding of Herceptin® to SKBR3 cells in a dose-dependent manner as described in Example 8.

FIG. 7: Omnitarg-Fab competitive FMAT. Dilutions of periplasmic extracts containing HER2 binding Nanobodies were tested for their ability to block the binding of Omnitarg-Fab (OT-Fab) to HER2-overexpressing SKBR3 cells as described in Example 9. (cells) represents the signal obtained for binding of biotinylated OT-Fab without addition of periplasmic extract. Addition of periplasmic extract containing a non-HER2 binding Nanobody (irr) had no influence on the binding of OT-Fab to SKBR3 cells. Periplasmic extracts 47A8, 47A11, 47B1, 47B12, 47D1, 47D4, 47D5, 47E7, 47F5 and 47G7 blocked binding of OT-Fab to HER2 with more than 85%.

FIG. 8: Growth inhibitory effect of monovalent HER2 binding Nanobodies on ErbB2-overexpressing SKBR3 cells. SKBR3 cells were seeded in 96 well plates and allowed to adhere as explained in Example 11. HER2-binding Nanobodies 5F7, 2A5, 2A4, 2D3 and 2C3, non-HER2 binding irrelevant Nanobody 12B2 or medium alone were added and the cells were incubated for 3 days. During the last 24 h, cells were pulsed with 1 μCi [³H]-thymidine. Incorporation of [³H]-thymidine was measured as described in Example 11.

FIG. 9: Herceptin®-competitive FMAT. Dilutions of monovalent, bivalent and bispecific Nanobodies were tested for their ability to block the binding of Herceptin® to HER2-overexpressing SKBR3 cells as described in Example 12. Bispecific Nanobodies 2A4-9GS-ALB1 and 2A5-9GS-ALB1 blocked the binding of Herceptin® to HER2-expressing SKBR3 cells to the same extent as the monovalent 2A4 and 2A5 Nanobodies respectively. Bivalent 2A4-9GS-2A4 and 2A5-9GS-2A5 Nanobodies blocked the binding of Herceptin® to HER2-expressing SKBR3 cells to a greater extent than their monovalent format. A: 2A4 derivatives; B: 2A5 derivatives.

FIG. 10: Design of biparatopic Nanobody expression vector as described in Example 13.1.

FIG. 11: SKBR3 cell proliferation assay with biparatopic Nanobodies purified from periplasmic extracts derived from plate 27 by PhyTip200⁺. Biparatopic Nanobodies 27A2-35GS-2D3, 27A5-35GS-2D3, 27B3-35GS-2D3, 27B5-35GS-2D3, 27C4-35GS-2D3, 27D3-35GS-2D3 and 27D6-35GS-2D3 block SKBR3 cell proliferation to a greater extent than 50 nM Herceptin®. Biparatopic Nanobodies 27A7-35GS-2D3, 27A9-35GS-2D3, 27A11-35GS-2D3, 27A12-35GS-2D3, 27B11-35GS-2D3, 27C11-35GS-2D3 and 27D7-35GS-2D3 display an agonistic effect.

FIG. 12: Sensorgram of monovalent 2D3, bivalent 2D3-35GS-2D3 and dummy-2D3 biparatopic Nanobodies.

FIG. 13: Sensorgram of monovalent 2D3 and biparatopic Nanobodies 27B7-35GS-2D3, 27C3-35GS-2D3 and 27H5-350S-2D3.

FIG. 14: Sensorgram of monovalent 2D3, bivalent 2D3-35GS-2D3 and biparatopic Nanobodies 27A3-35GS-2D3, 27E7-35GS-2D3 and 27D1-35GS-2D3.

FIG. 15: Herceptin®-competitive FMAT. Dilutions of monovalent 2D3, bivalent 2D3-35GS-2D3 and biparatopic Nanobodies combining the Herceptin®-competitive 2D3 and a HER2-binding or dummy Nanobody were tested for their ability to block the binding of Herceptin® to HER2-overexpressing SKBR3 cells as described in Example 14.2. A: Bivalent 2D3-35GS-2D3 and biparatopic Nanobodies 27H3-35GS-2D3 and 27D1-35GS-2D3 block binding of Herceptin® to HER2 expressed on SKBR3 cells more efficiently than monovalent 2D3 Nanobody. B: Nanobodies 27A3, 27A5 and 30D10 have no influence on the Herceptin®-competitive behavior of Nanobody 2D3 when fused to its N-terminal end, spaced by a 35GS linker. C: Nanobodies 27B7, 27C3, 27H5 and the dummy Nanobody have an inhibitory effect on the Herceptin®-competitive potential of 2D3 when fused to its N-terminal end, spaced by a 35GS linker.

FIG. 16: Omnitarg-Fab competitive FMAT. Dilutions of OT-Fab, monovalent 2D3 and biparatopic Nanobodies 27C3-35GS-2D3, 27A5-35GS-2D3, 27H3-35GS-2D3 and dummy-35GS-2D3 were tested for their ability to block the binding of OT-Fab to HER2-overexpressing SKBR3 cells as described in Example 14.3. None of the bipartope Nanobodies, nor monovalent 2D3 blocked the binding of OT-Fab to HER2 expressed on SKBR3 cells. OT-Fab blocked binding of biotinylated OT-Fab in a dose-dependent manner.

FIG. 17: Effect of biparatopic Nanobodies on SKBR3 tumor cell proliferation. Biparatopic Nanobodies 27A5-35GS-2D3, 27A3-35GS-2D3 and 30D10-35GS-2D3 significantly block proliferation of SKBR3 tumor cells and to a greater extent than the monovalent 2D3 and dummy-2D3 biparatopic Nanobody.

FIG. 18: Effect of biparatopic Nanobodies on AKT signaling in SKBR3 cells (see Example 16). Biparatopic 27A3-35GS-2D3 and 27A5-35GS-2D3, Herceptin®, but not dummy-2D3 biparatopic or monovalent 2D3 Nanobody inhibits AKT phosphorylation in whole SKBR3 cell lysates.

FIG. 19: Sensorgram of HER2-ECD binding to 2D3, 47D5 or the biparatopic Nanobody 2D3-35GS-47D5.

FIG. 20: Effect of biparatopic Nanobodies combining Herceptin®-competitive and Omnitarg competitive Nanobodies, monovalent Nanobodies 2D3, 5F7 and 47D5, Omnitarg-Fab and Herceptin® on HRG-mediated activation of mitogen-activated protein kinase (MAPK).

FIG. 21: Effect of biparatopic Nanobodies combining Herceptin®-competitive and Omnitarg competitive Nanobodies, monovalent Nanobodies 2D3, 5F7 and 47D5, Omnitarg-Fab and Herceptin® on HRG-mediated activation of Akt signaling.

FIGS. 22A and 22B: Model of NB-2D3 (indicated) linked to another Nanobody (indicated) docked on HER-2. The linker is indicated as well. N denotes the N-terminus of NB-2D3; C is the C-terminus of Nb-2D3.

FIG. 23: Energy penalty values for each residue in the linker +/−10 residues of each Nanobody connected to the linker in the biparatopic construct 5F7-35GS-47D5 with appropriate linker length. None of the residues of the linker or at the connection points of the linker with the Nanobodies (NB-1 and NB-2) have a high energy penalty value.

FIG. 24: Energy penalty values for each residue in the linker +/−10 residues of each Nanobody connected to the linker in the biparatopic construct 47D5-35GS-5F7 with unappropriate linker length. High energy penalty values are observed at the C-terminal connection of the linker with the N-terminal end of the second Nanobody (NB-2).

FIG. 25: Energy penalty values for each residue in the linker +/−10 residues of each Nanobody connected to the linker in a biparatopic construct with the same Nanobodies as in FIG. 23 but with a longer linker length (47D5-40GS-5F7). We see that the high energy penalty values at the connection of the C-terminal end of the linker with the N-terminal end of NB-2 are reduced suggesting a more appropriate linker length. The energy penalty values at both ends of the linker are still higher than those observed in FIG. 22, indicating a still not optimal linker.

FIG. 26A: Backbone RMSD (Å²) between the 5F7-linker-47D5 constructs (built by homology modelling) with the individual Nanobodies 5F7 and 47D5 in their unlinked binding mode. The linker length varies from 5 to 35.

FIG. 26B: Ribbon view of the 5F7-linker-47D5 biparatopic construct for 2 linker lengths. The binding mode of the individual Nanobodies and the biparatopic constructs are shown. The HER-2 target is omitted for clarity. On the left side: a 35GS linker is used between the 2 Nanobodies and a very limited deviation from the individual binding modes is observed. On the right side: a 5GS linker is used and it can clearly been observed that both Nanobodies in the biparatopic construct significantly deviate from their optimal binding mode.

FIGS. 27A-K: Figures illustrating some of the preferred aspects and some of the advantages of the present invention, including the multiparatopic polypeptides of the invention.

EXAMPLES Example 1 Procurement of the Extracellular Domain of HER2 for Use as Selection Antigen in Phage Display

1.1 Cloning of Extracellular HER2 Domain

cDNA was isolated from SKBR3 breast cancer cells. The isolation of total RNA and cDNA synthesis was done according to standard protocols (Sambrook, Molecular cloning: Laboratory manual, 2^(nd) edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The coding sequence of the extracellular domain of the HER2 antigen was amplified by PCR using primer For-ErbB2 ECD; GCGAGCACCCAAGTGTGCACC (SEQ ID NO: 2392) and primer Rev-ErbB2 ECD: CTGCTCGGCGGGGCAGCCCTT (SEQ ID NO: 2393). The PCR construct was then cloned into the pCR4-TOPO cloning vector (Invitrogen, Paisley, UK). Clone 4 having the correct sequence was then amplified by PCR using primers (For-pST ErbB2 ECD: GGCGCGCCGACTACAAAGACGATGACGACAAGAGCACCCAAGTGTGCACC (SEQ ID NO: 2394) and Rev-pST ErbB2 ECD: CGGCTCGAGCTATTAATGAGAATGGTGATGGTGCTCGGCGGGGCAGCCCTT (SEQ ID NO: 2395)) that were designed to introduce restriction sites at the beginning and the end of the fragment encoding the HER2-ECD. The PCR product was then cloned via AscI and XhoI into the plasmid pSecTag-HygroA (Invitrogen, Paisley, UK). As such, the coding sequence of the HER2-ECD was fused in frame with the Ig-κ chain leader sequence at its N-terminal end followed by a Flag tag and a polyhistidine tag at the C-terminus. The sequence of different clones was determined by sequencing according to standard protocols.

1.2 Expression of the Extracellular Domain of HER2 in HEK 293 Cells, Purification of the Recombinant Protein

Expression of the extracellular domain of HER2 was performed in HEK293T cells. HEK293T cells were seeded at 2×10⁶ cells in 20 ml Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS in T75 tissue culture flasks and allowed to adhere overnight. The next day, culture supernatant was removed and the cells were transiently transfected with purified pSecTag-HygroA plasmid DNA using Fugene-HD (Roche, Basel, Switzerland) as transfection agent. Cells were grown for an additional 72 h in DMEM containing 0.1% FBS, after which the culture supernatant was collected and filter-sterilized on a 0.22 μm filter (Millipore). The construct was then further purified out of the culture supernatant by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC).

Detection of the recombinant protein was performed by ELISA. Maxisorp 96-well plate (Nunc, Wiesbaden, Germany) was coated with an anti-flag monoclonal antibody (Sigma Aldrich, Bornem, Belgium). Unspecific binding was blocked with 2% milk powder in PBS for 2 hours. All prior and subsequent washes were performed with PBS. Afterward, eluate fractions were incubated for 2 hours at room temperature, followed by incubation with Herceptin®. Detection of the recombinant HER2-ECD was performed with a horseradish peroxidase conjugated anti-IgG antibody (Jackson Immunoresearch Laboratories, Suffolk, UK). Development of the ELISA was performed with TMB substrate (Pierce, Rockford, Ill.) according to the specifications of the manufacturer

Example 2 Procurement of Omnitarg-Fab for Use as Competitive Agent in Phage Display and Screening Assays

2.1 Cloning of Omnitarg-Fab

Omnitarg-Fab was constructed by gene assembly. The amino acid sequence of variable light and variable heavy chain of Omnitarg was derived from patents WO 2006/044908 and WO 2004/048525. The sequence was backtranslated and codon optimized using Leto 1.0 Gene optimization software (www.entechelon.com). Oligonucleotide primers for assembly of the variable light chain (V_(L)), variable heavy chain (V_(H)), constant light chain (C_(L)) and constant domain 1 of the heavy chain (CH₁) of the Omnitarg-Fab were designed (Tables C-5 and C-6) and assembly PCR performed. The introduced restriction sites SfiI and BsiWI for the V_(L), KpnI and BstEII for the V_(H), BsiWI and AscI for the C_(L), and BstEII and NotI for the CH₁ were utilized for sequential cloning into an in-house expression vector derived from pUC119 which contained the LacZ promoter, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the Omnitarg-Fab coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag. Oligonucleotide sequences were designed to have a 15 nucleotide overlap with 5′ and 3′ overlapping oligonucleotides. Three consecutive PCR overlap extension rounds were performed using Expand High fidelity PCR system (Roche, Basel, Switzerland) to obtain V_(L), V_(H), C_(L) and CH₁ respectively. The obtained PCR fragments were cloned into the pCR4-TOPO cloning vector (Invitrogen, Paisley, UK). Plasmid. DNA was prepared from clones having the correct sequence. The fragments were isolated from the pCR4-TOPO cloning vector via restriction with the appropriate enzymes and extraction of the fragments from agarose gel. The fragments were then consecutively cloned into the in-house expression vector.

2.2 Expression of the Omnitarg-Fab in E. coli Cells, Purification of the Recombinant Protein

The Omnitarg-Fab fragment was expressed in E. coli as His6-tagged protein and subsequently purified from the culture medium by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC).

Omnitarg-Fab was biotinylated using EZ-Link Sulpho-NHS-LC-Biotin labeling kit according to the manufacturer's instructions (Pierce, Rockford, Ill.). Removal of free biotin was performed on Zeba Desalt Spin columns according to the manufacturer's instructions (Pierce, Rockford, Ill.).

Example 3 Identification of HER2 Binding Nanobodies

3.1 Immunizations

After approval of the Ethical Committee of the Faculty of Veterinary Medicine (University Ghent, Belgium), 2 llamas (121, 122) were immunized, according to standard protocols, with 6 intramuscular injections at biweekly intervals of SKBR3 human tumor cells which are derived from a breast tumor and contain an amplified HER2 gene and overexpress HER2 p185 tyrosine kinase (SKBR3; ATCC HTB-30; LGC Promochem, Middlesex, UK). Each dose consisted of approximately 5×10⁷ freshly harvested SKBR3 cells.

3.2 Evaluation of Induced Responses in Llama

At day 0, 42 and 81 (time of PBL collection), sera were collected to evaluate the induction of immune responses in the animals against HER2 by ELISA. In short, 2 μg/ml recombinant human ErbB2/Fc chimera (rhErb2-Fc; R&D Systems, Minneapolis, Minn.) were immobilized overnight at 4° C. in a 96 well Maxisorp plate (Nuns, Wiesbaden, Germany). Wells were blocked with a casein solution (1% in PBS). After addition of serum dilutions, specifically bound immunoglobulins were detected using a goat anti-llama horseradish peroxidase conjugate (Bethyl Lab. Inc., Montgomery, Tex.), showing that for all animals a significant antibody dependent immune response against HER2 was induced (FIG. 1A). The antibody response was mounted both by the conventional and the heavy chain only antibody expressing B-cell repertoires since specifically bound immunoglobulins could be detected with antibodies specifically recognizing the conventional llama IgG1 antibodies (FIG. 1B) or the heavy-chain only llama IgG2 (FIG. 1C) and IgG3 (FIG. 1D) antibodies.

3.3 Library Construction

When an appropriate immune response was induced in llama, four days after the last antigen injection, a 150 ml blood sample was collected and peripheral blood lymphocytes (PBLs) were purified by a density gradient centrifugation on Ficoll-Paque™ (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions. Next, total RNA was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into a phagemid vector derived from pUC119 which contained the LacZ promoter, a coliphage pill protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use.

3.4 Selections

Phage libraries obtained from llamas 121 and 122 were used for different selections.

In a first selection, ErbB2/Fc chimera (R&D Systems, Minneapolis, Minn., US) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 20, 5 and 1 nM. Following incubation with the phage libraries and extensive washing, bound phage was aspecifically eluted with trypsin (1 mg/ml).

In a second selection, ErbB2/Fc chimera (R&D Systems, Minneapolis, US) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 20 nM. Following incubation with the phage libraries and extensive washing, bound phage was specifically eluted with Herceptin® (Genentech, Roche).

In a third selection, soluble biotinylated ErbB2/Fc chimera was incubated with the phage libraries. After extensive washing, the biotinylated ErbB2/Fc was captured on a neutravidin coated solid phase. Bound phage was aspecifically eluted with trypsin (1 mg/ml).

In a fourth selection, soluble biotinylated ErbB2/Fc chimera was incubated with the phage libraries. After adding a 100-fold excess of non-labeled HER2, the biotinylated ErbB2/Fc was captured on a neutravidin coated solid phase. Bound phage was aspecifically eluted with trypsin (1 mg/ml).

In a fifth selection, phage libraries were incubated with Herceptin®-captured ErbB2/Fc. After extensive washing, bound phage was aspecifically eluted with trypsin (1 mg/ml)

In a sixth selection, soluble biotinylated ErbB2/Fc chimera was incubated with the phage libraries. After extensive washing, the biotinylated ErbB2/Fc was captured on a neutravidin coated solid phase. Bound phage was specifically eluted with Omnitarg-Fab.

In a seventh selection, phage libraries were incubated with Herceptin®-captured ErbB2/Fc. After extensive washing, bound phage was specifically eluted with Omnitarg-Fab.

In an eighth selection, phage libraries were incubated with biotinylated extracellular domain of HER2 captured on a neutravidin coated solid phase. After extensive washing, bound phage was specifically eluted with Omnitarg-Fab.

In a ninth selection, phage libraries were incubated with biotinylated extracellular domain of HER2 captured on a neutravidin coated solid phase. After extensive washing, bound phage was specifically eluted with Herceptin®.

In all selections, enrichment was observed. The output from each selection was recloned as a pool into an expression vector derived from pUC119 which contained the LacZ promoter, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag. Colonies were picked and grown in 96 deep-well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 4 Detection and Isolation of HER2-Specific Heavy Chain Antibody Producing B-Cells

PBMC were isolated from peripheral blood samples from llamas immunized with HER2-Fc or SKBR3 human tumor cells using Ficoll density gradient centrifugation. These were then resuspended in cell culture medium and partially depleted from monocytes by adherence to the surface of plastic tissue culture T-flasks.

Next, non-adherent PBMC were collected from the flasks, washed with FACS buffer (PBS/10% FCS) at 4° C. and resuspended in the same ice-cold buffer. These were then stained using a combination of Alexa 488 labeled HER2-Fc (produced in-house, using Invitrogen (Paisley, UK) activated Alexa 488 and HER2-Fc recombinant protein from R&D Systems (Minneapolis, Minn.)), phycoerythrin labeled mouse-anti-llama IgG2 and -3 monoclonal antibodies (produced in-house, using purified phycoerythrin from Cyanotech, (Kailua-Kona, Hi.) crosslinked using the sulfo-SMCC heterobifunctional linker from Pierce-Endogen (Rochford, Ill.) to in-house produced and purified monoclonal antibodies originally described in Daley et al. (Clin. Diagn. Lab. Immunol. 2005, 12: 380)), Alexa 647 labeled mouse-anti-llama IgG1 monoclonal antibody (produced in-house), Alexa 647 labeled mouse-anti-llama monocyte and neutrophil antibody DH59B (purified antibody obtained from VMRD Inc. (Pullman, Wash.)) and dead cell specific dye TOPRO3 (Invitrogen, Paisley, UK). In some experiments, in-house Alexa 647 labeled recombinant human IgG1 Fc fragment (R&D Systems, Minneapolis, Minn.) was added to the stain combination as well.

Stained samples were washed thoroughly using cold FACS buffer and analyzed on a standard two-laser BD FACSAria cell sorter equipped with the ACDU microtiter plate single-cell deposition option (BD Biosciences, Franklin Lakes, N.J.). During acquisition and analysis, a gate was set on lymphocytes based on their forward/side scatter profile, which overlaps considerably with monocytes in llama. Doublet events were eliminated from acquisition and analysis by forward as well as side scatter pulse processing, eliminating all events which might be originating from more than one cell. Dead cells, monocytes and B-cells expressing conventional antibody on their cell membrane were removed from further analysis by gating out all remaining events having fluorescence over background (unstained PBMC) in the Alexa647/TOPRO3 channel. In some experiments, Alexa 647 labeled recombinant Fc fragment was used to stain the PBMC additionally. In these experiments, B-cells producing antibody binding Fc were also rejected from analysis and sorting by similar Alexa 647 channel exclusion, so as to avoid isolation of B-cells binding the Fc region of the fusion protein. In the phycoerythrin channel. B-cells displaying heavy chain antibody on their cell membrane could be clearly differentiated from any other remaining lymphocyte-type cells, and another gate was set on this population. Lastly, antigen-binding heavy chain IgG expressing B-cells cells were detected as a discrete high fluorescence intensity peak population in the Alexa 488 channel histogram distinct from the main population being no more fluorescent in this channel than when no Alexa 488 labeled antigen was added. Individual antigen binding B-cells were collected in separate wells of 96-well PCR plates in the ACDU, using DiVa software predefined stringent single-cell sorting criteria to avoid any double-cell droplet or adjacent-droplet double cell sorting. Typically, only 1-5% of heavy chain B-cells were found to bind antigen.

Example 5 Amplification and Cloning of HER2-Specific Heavy Chain Antibody Variable Regions

Individual B-cells expressing heavy chain antibodies binding HER2-Fc or the HER2 region of the fusion protein specifically were sorted into 96-well plates containing 40 μl of RT-PCR buffer (Superscript III One-step RT-PCR kit, Invitrogen, Paisley, UK) per well, as described in Example 5, and stored at −80° C. For variable region gene sequence recovery, plates were thawed at room temperature and a mix of NP-40 (Roche Applied Sciences, Indianapolis, Ind.), gene specific 5′ and 3′ primers and RT-PCR enzyme mix were added to a total volume of 50 microliter per well by an automated liquid handler (Tecan, Männedorf, Switzerland). After reverse transcription and first PCR amplification in a standard thermal cycler, a 2 microliter aliquot was removed from all wells and amplified in a nested PCR reaction using a proof-reading thermostable polymerase, or blend of polymerases containing at least one proof-reading enzyme. The 5′ nested primer contains the nucleotide sequence required for directional TOPO cloning (Invitrogen, Paisley, UK). The 3′ primer is designed to allow for the in-frame fusion of variable region gene framework 4 to vector encoded detection (c-myc) and purification (6His) peptide tags. Amplicons were detected from individual wells using ethidium bromide stained agarose gels and/or in microtiter plates via PicoGreen DNA binding fluorescent dye assay (Invitrogen, Paisley, UK). Typically, up to 60% of wells contained a single and sharply defined amplification product, whereas control wells in the same plate not having received any cells were completely devoid of amplification product.

The amplicons from nested PCR wells containing detectable product were then ligated into an E. coli expression vector in a homogenous ligation reaction, by mixing an aliquot of unpurified PCR mix with a topoisomerase-activated expression vector (in-house developed IPTG inducible E. coli Nanobody expression vector, adapted to allow directional TOPO cloning by Invitrogen's custom services department). The ligation mixture was then pipetted onto electrocompetent E. coli cells pre-aliquotted in a 96-well format electroporation chamber array (BTX Products of Harvard Apparatus, Holliston, Mass.), and cells were transformed by electroporation using a BTX pulse generator.

Transformation mix was spread on selective agarose, multiple individual subcolonies picked and grown in 96-well deep well plates containing liquid selective medium by a QP Expression colony picker/rearrayer system (Genetix, New Milton, Hampshire, UK).

Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 6 Anti-HER2 Nanobodies Recognize Extracellular HER2 Domain

Periplasmic extracts of individual Nanobodies were screened for HER2 specificity by ELISA on solid phase coated ErbB2/Fc chimera (R&D Systems, Minneapolis, Minn.). Detection of Nanobody fragments bound to immobilized recombinant HER2 antigen was carried out using an in house made mouse anti-myc antibody (2 mg/ml) detected with alkaline phosphatase-conjugated anti-mouse IgG (Sigma Aldrich, Bornem, Belgium). The signal was developed by adding PNPP substrate solution and detected at a wavelength of 405 nm. FIG. 2 is illustrative of typical ELISA results, showing a high hit rate of positive clones.

Sequences of different HER2 binding clones are depicted in Tables B-1, B-2 and B-3. Alignment of the different HER2 binding clones based on CDR3 similarity is depicted in Table C-1.

Example 7 Anti-HER2 Nanobodies Recognize Cell Surface Exposed Receptor Epitopes

To verify whether the Nanobodies are able to recognize cell surface expressed HER2, binding to breast cancer tumor cell line SKBR3 was assessed by flow cytometry. Cell binding assays were carried out by initially incubating 200,000 cells with Nanobody-containing periplasmic preparation obtained in Examples 3 and 5 or relevant controls. After incubation, the cells were washed with FACS buffer. Cells were subsequently incubated successively with an in-house mouse anti-myc-tag monoclonal antibody and phycoerythrin labeled goat anti-mouse F(ab′)₂ fragments (Jackson ImmunoResearch, Suffolk, UK). To omit signals arising from dead cells, a TOPRO-3 (Invitrogen, Paisley, UK) staining was carried out. Cells were finally analyzed on a BD FACSArray Bioanalyzer System (BD Biosciences, Franklin Lakes, N.J., US).

FIG. 3 depicts binding of several Nanobody constructs to SKBR3 cells as measured by flow cytometric analysis. It can be seen that the constructs 2A1, 2A3, 2C3, 2C5, 2D3 and 2G4 show clearly discernable shifts in fluorescence intensity as compared to the fluorescence intensity for cells incubated only with FACS buffer in the absence of any construct but with all appropriate detection agents as used for the detection of Nanobody constructs.

Example 8 Screening for Nanobodies that Compete with Herceptin® for HER2 Binding

A competition ELISA was performed to screen for Nanobodies that are able to inhibit the Herceptin® interaction with HER2. In this competition ELISA, the binding of 2 nM Herceptin® to SKBR3 vesicles was evaluated in the presence of a 1/20 dilution of Nanobody containing periplasmic extract obtained in the second selection described in Example 3. FIG. 4 shows an example of this competitive ELISA, identifying several clones that compete with binding of Herceptin® to HER2 expressed on SKBR3 vesicles.

Periplasmic extracts obtained in the second and ninth selection described in Example 3 and periplasmic extracts obtained in Example 5, were also screened in a Herceptin®-competitive homogeneous cell-based assay to evaluate the capacity of the expressed Nanobodies to block Herceptin® binding to HER2. The FMAT 8200 HTS system (Applied Biosystems, Foster City, Calif.) assay was performed as follows: SKBR3 cells expressing HER2 were grown in tissue culture flasks, collected and washed with screening buffer (PBS, 10% FCS) and resuspended in screening buffer at a concentration of 2.5×10⁵ cells/ml. Alexa 647-labeled Herceptin® was diluted to 62.5 ng/ml in screening buffer. Periplasmic extracts were diluted in screening buffer to obtain final dilutions of 4, 10, 40, 100, 200 and 400. To initiate the competitive screen, 10 μl labeled Herceptin®, 10 μl periplasmic dilution and 20 μl of cells were added to each well of FMAT system 384-well plates (PE Biosystems, Foster City, Calif.) The plates were scanned after 2 hours of incubation. A well was considered positive if it had a count of over 50 events. Screening of the extracts in this Herceptin® competitive homogeneous cell-based assay identified several clones (SEQ ID NOs: 2051-2113) that can block the binding of Herceptin® to HER2 with more than 90% (FIG. 5).

Purified Nanobodies were tested for inhibition of binding of Alexa647-labeled Herceptin® to HER2 expressed on SKBR3 cells. Serial dilutions of purified Nanobody (concentration range: 20 nM-10 pM) were added to SKBR3 cells together with 4×10⁻¹⁰M Alexa647-labeled Herceptin® and incubated for 2 hours, after which plates were scanned. Herceptin® was included as reference (MoAb). Results are shown in FIG. 6. Dose-response curves were observed for all Nanobodies with IC₅₀-values ranging from 40 pM to 200 pM.

Example 9 Screening for Nanobodies that Compete with Omnitarg-Fab for HER2 Binding

Periplasmic extracts obtained in the sixth and seventh selection described in Example 3, were screened in an Omnitarg-Fab (OT-Fab) competitive homogeneous cell-based assay to evaluate the capacity of the expressed Nanobodies to block OT-Fab binding to HER2. The FMAT 8200 HTS system (Applied Biosystems, Foster City, Calif.) assay was performed as follows: SKBR3 cells expressing HER2 were grown in tissue culture flasks, collected and washed with screening buffer (PBS, 10% FCS) and resuspended in screening buffer at a concentration of 2.5×10⁵ cells/ml. Biotinylated OT-Fab was diluted in screening buffer to obtain a final concentration of 0.586 nM. The periplasmic extracts were diluted in screening buffer to obtain final dilutions of 100. To initiate the competitive screen, 5 μl labeled OT-Fab, 10 μl periplasmic dilution, 5 μl. FMAT Blue dye-labeled streptavidin (100 ng/ml) and 20 μl of cells were added to each well of FMAT system 384-well plates (PE Biosystems, Foster City, Calif.). The plates were scanned after 2 hours of incubation. A well was considered positive if it had a count of over 50 events. Screening of the extracts in this OT-Fab competitive homogeneous cell-based assay identified clones that can block the binding of OT-Fab to HER2 with more than >90% (FIG. 7). Sequence analysis showed that all clones that blocked binding of OT-Fab HER2 are identical and represent a single Nanobody (SEQ ID NO: 2114).

Example 10 Screening of Kinetic Off-Rate Constants Via Surface Plasmon Resonance (BIAcore)

RhErbB2-Fc was immobilized on a CM5 sensor chip surface docked in Biacore 3000. Approximately 3600RU of rhErbB2-Fc was immobilized. Experiments were performed at 25° C. Periplasmic extracts were diluted 10-fold in running buffer (HBS-EP). The samples were injected for 1 min at a flow rate of 45 μl/min over the activated and reference surfaces. Those surfaces were regenerated with a 3 s pulse of glycine-HCl μl-11.5+0.1% P20. As an example, the off rate (k_(off)) of different Nanobodies is documented in Table C-2.

Example 11 Anti-HER2 Nanobodies can Block SKBR3 Cell Proliferation

The growth inhibitory characteristics of isolated Nanobodies were evaluated using the breast tumor cell line SKBR3. Briefly, SKBR3 cells were detached using 0.25% (vol/vol) trypsin and suspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS), glutamine, and penicillin-streptomycin at a density of 1×10⁵ cells/ml. Aliquots of 200 μl (2×10⁴ cells) were plated into 96-well microdilution plates and allowed to adhere. After overnight adherence, cells were washed with serum-free medium and starved for 4 hours in 100 μl serum-free medium. Then, 100 μl of 1% FCS containing medium alone or medium containing Nanobody (final concentration of 50 nM) was added. After 2 days of incubation, cells were pulsed with 1 μCi [³H]-thymidine and incubated for an additional 24 h prior to freezing at −80° C. Cells were subsequently thawed and embedded on glass fiber membranes using a cell harvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA). After several washings with water, filters were air-dried and counted using a γ-counter (Perkin Elmer Life Sciences). Nanobody 2A5 inhibited SKBR3 proliferation by about 18%. Up to 30% or more inhibition was achieved with Nanobodies 2C3, 2D3, 2A4 and 5F7 (FIG. 8).

Example 12 Generation of Multivalent/Multispecific Nanobody Formats

To potentially increase the biological effect of Nanobody molecules, bivalent constructs were fused head-to-tail using a GGGGSGGGS linker.

Here we describe the construction and characterization of bivalent Nanobodies consisting of two identical anti-HER2 molecules all separated by a 9 (GS) amino acid linker peptide. DNA segments encoding Nanobodies 2A4, 2A5, 2C3, 2D3, 5F7 were head-to-tail fused resulting in constructs 2A4-9GS-2A4, 2A5-9GS-2A5, 2C₃₋₉GS-2C3, 2D3-9GS-2D3, 5F7-9GS-5F7. Sequences of these bivalent constructs are listed in Table B-4. All Nanobodies were expressed in E. coli and purified according to standard protocols (see for example the prior art and applications filed by applicant cited herein).

The different bivalent Nanobody formats were screened in a Herceptin®-competitive homogeneous cell-based assay to evaluate their capacity to block Herceptin® binding to HER2 compared to their monovalent format. Briefly, 10 μl labeled Herceptin® (62.5 ng/ml), 10 μl Nanobody dilution and 20 μl of cells (5×10³ cells) were added to each well of FMAT system 384-well plates (PE Biosystems, Foster City, Calif.). The plates were scanned after 2 hours of incubation. FIG. 9 shows that the bivalent constructs are more efficient in blocking the binding of Herceptin® to HER2-expressing SKBR3 cells as compared to their monovalent formats.

To test whether selected Nanobodies have potential as anticancer agents in an animal model, a strategy to increase the serum half life is preferred (as for example described in patent application WO 04/041865), since the serum half life of a mono- or bivalent Nanobody (approximately 15 or 30 KDa, respectively) is not optimal for this therapeutic indication. Human serum albumin specific Nanobody ALB1 (SEQ ID NO: 2391), cross reactive with mouse serum albumin, was chosen. Here we describe the construction of bispecific Nanobodies consisting an anti-HER2 Nanobody and ALB1, all separated by a 9 (GS) amino acid linker peptide and resulting in constructs 2A4-9GS-ALB1, 2A5-9GS-ALB1, 2C3-9GS-ALB1, 2D3-9GS-ALB1 and 5F7-9GS-ALB1. Sequences of these bispecific constructs are given in Table B-5.

To test whether the HER2-binding Nanobodies as disclosed herein above retain their biological activity in a more complicated molecular context such as a bispecific format, Nanobody formats were screened in a Herceptin®-competitive homogeneous cell-based assay to evaluate their capacity to block Herceptin® binding to HER2 compared to their monovalent and bivalent format. Based on the results shown in FIG. 9, it can be concluded that fusion of a Nanobody with different antigen specificity to a HER2-binding Nanobody does not affect the potency of the latter.

Example 13 Generation of Biparatopic Formats Combining a Herceptin®-Competing Nanobody with a Library of HER2 Binding Nanobodies

The structural requirement for multispecificity is to fuse two or more binding domains together, with sufficient flexibility to allow simultaneous binding to different target epitopes. The simplest bispecific is one that binds to two different and non-overlapping epitopes on the same target in such a way that simultaneous binding to the target is possible. Robert et al (Int. J. Cancer 1995, 28; 62(3): 283-90) have described the design of high avidity biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen. Binding of both arms simultaneously without a significant loss of entropy will endow ‘biparatopic’ antibodies with increased avidity and hence, increased binding affinity to the target. As a result, higher potency can be obtained as well as enhanced selectivity. In addition, careful selection of the epitopes targeted on the antigen by the biparatopic antibody or fragment thereof, combined with rational design of linkers to allow maximal flexibility of the two binding domains within the biparatopic antibody, may for example result in the blocking of two or more critical interaction sites of the target, leading to improved potency.

Using genetic fusion, one Herceptin®-competing Nanobody was combined with a repertoire of HER2-binding Nanobodies and this mini-repertoire was screened for biparatopics with improved binding activity and tumor cell growth inhibitory characteristics compared to the monovalent Herceptin®-competing Nanobody.

13.1 Construction of an Expression Vector for Biparatopic Design

For the construction of biparatope Nanobodies, an expression vector was adapted to contain the Herceptin®-competitive Nanobody 2D3 (which was shown to block cell proliferation between 20-30% as monovalent format (see Example 11) and which strongly competes with Herceptin® for binding to HER2-overexpressing SKBR3 cells) to which other Nanobodies with different HER2-binding specificities can be fused, spaced by a linker (FIG. 10). For the design of this vector, a 35 GS linker was used but other linker lengths can also be used to allow flexibility between the two building blocks. The 2D3 Nanobody is placed at the C-terminal end of the construct to allow SfiI-BstEII cloning of a full selection output. Alternatively, the 2D3 Nanobody can also be placed at the N-terminal end of the construct to allow cloning of a full selection output at the C-terminal end.

13.2 Generation of a Biparatopic Library

A full selection output retrieved from a selection on Herceptin®-captured rhErbB2/Fc followed by trypsin elution (Example 3.4), was unidirectionally cloned to the 2D3 Nanobody. Sequence analysis of a selected number of individual colonies derived from the selection output showed a good diversity in the repertoire: 16 Nanobody families were identified in 72 sequences. The ligation mix was transformed into E. coli cells and the transformation mix spread on selective agarose. Multiple individual subcolonies were picked and grown in 96-well deep well plates containing liquid selective medium by a QP Expression colony picker/rearrayer system (Genetix, New Milton, Hampshire, UK). Forty-eight individual colonies were sequenced and analyzed. From 32 annotated sequenced, eight different Nanobody families were identified.

Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). The biparatopic Nanobodies were purified from the periplasmic extracts using PhyTip200⁺ columns (Phynexus, San Jose, Calif.) by a Tecan Evo Robotic system (Promega, Madison, US) and analyzed for their effects on SKBR3 tumor cell proliferation.

13.3 Effect of Biparatopic Nanobodies on SKBR3 Cell Proliferation

The growth inhibitory characteristics of Nanobodies purified from periplasmic extracts by PhyTip200⁺ were evaluated using the breast tumor cell line SKBR3. Briefly, SKBR3 cells were detached using 0.25% (vol/vol) trypsin and suspended in DMEM supplemented with 10% fetal calf serum (FCS), glutamine, and penicillin-streptomycin at a density of 1×10⁵ cells/ml. Aliquots of 200 μl (2×10⁴ cells) were plated into 96-well microdilution plates and allowed to adhere. After overnight adherence, cells were washed with serum-free medium and starved for 4 hours in 100 μl serum-free medium. Then, 100 μl of 1% FCS containing medium alone or 90 ml of 1% FCS containing medium with 10 μl PhyTip200⁺ purified periplasmic extract or 50 nM Herceptin® was added. After 2 days of incubation, cells were pulsed with 1 μCi [³H]-thymidine and incubated for an additional 24 h prior to freezing at −80° C. Cells were subsequently thawed and embedded on glass fiber membranes using a cell harvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA). After several washings with water, filters were air-dried and counted using a γ-counter (Perkin Elmer Life Sciences).

Herceptin® was able to inhibit cell proliferation of SKBR3 up to 50%. Different subclasses of biparatopic Nanobodies were identified: a group of biparatopic Nanobodies revealed an inhibitory effect on the ErbB2 overexpressing cell line SKBR3 to a lower extent than Herceptin®, a second group of biparatopic Nanobodies increased cell proliferation and a third group of biparatopic Nanobodies was able to inhibit cell proliferation of SKBR3 cells to an equal or greater extent than Herceptin®. FIG. 11 shows an example of this ‘single hit’ cell proliferation assay.

Example 14 Characterization of Biparatopic Nanobodies

The biparatopic molecules 28F6-35GS-2D3, 28G5-35GS-2D3, 29E9-35GS-2D3, 30D10-35GS-2D3, 27A5-35GS-2D3, 31D11-35GS-2D3, 30E10-35GS-2D3, 27A3-35GS-2D3, 27B7-35GS-2D3, 27C3-35GS-2D3, 27D1-35GS-2D3, 27E4-35GS-2D3, 27E7-35GS-2D3, 27H3-35GS-2D3, 27H4-35GS-2D3, 27H5-35GS-2D3 were expressed in E. coli as c-myc, His6-tagged proteins and subsequently purified from the culture medium by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). A control biparatopic Nanobody consisting of a dummy (i.e. not binding to HER2) Nanobody genetically fused to the 2D3 Nanobody, spaced by a 35GS linker was used as a control.

14.1 Biparatopic Nanobodies Display Improved Binding to HER2 as Compared to the Monovalent Building Blocks

The off-rate of the biparatopic Nanobodies was determined by surface plasmon resonance on a Biacore 3000 instrument. In brief, rhErbB2-Fc was immobilized on a CM5 sensor chip surface docked in Biacore 3000. Approximately 3600RU of rhErb B2-Fc was immobilized. Experiments were performed at 25° C. Nanobody binding was assessed at various concentrations. The samples were injected for 1 min at a flow rate of 45 μl/min over the activated and reference surfaces to allow for binding to chip-hound antigen. Next, binding buffer without Nanobody was sent over the chip at the same flow rate to allow for dissociation of bound Nanobody. After 10 min, remaining bound analyte was removed by injecting regeneration solution (Glycine/HCl pH1.5).

The monovalent 2D3 and biparatopic dummy-2D3 Nanobodies had similar off-rates in the range of 1E-3 l/s, indicating that fusion of a Nanobody to the N-terminal end of 2D3 does not interfere with binding of the latter (FIG. 12). The off-rate of bivalent 2D3-35GS-2D3 is in the range of 1E-4 l/s, indicating simultaneous binding of the two Nanobodies.

The off-rate of the biparatopic constructs 2B7-350S-2D3, 27C3-35GS-2D3 and 27H5-35GS-2D3 are in the range of 1E-3 l/s (FIG. 13). These off-rates and the binding responses indicate binding by the 2D3 paratope, but lack of binding by the other paratope, either by non-specificity for rhErb2 or an extremely much lower affinity for rhErb2 compared to 2D3 or by sterical hindrance of the epitope by the Fc part or by the altered conformation after the immobilization procedure on the CM5 sensor chip.

Off-rates of the biparatopic constructs 2D3-35GS-2D3, 27D1-35GS-2D3, 27A3-35GS-2D3, 27E7-35GS-2D3 are in the range of 1E-4 l/s (FIG. 14). These off-rates indicate simultaneous binding of the 2 paratopes.

14.2 Herceptin®-Competitive Behavior of Biparatopic Nanobodies

Biparatopic Nanobodies were screened in a Herceptin®-competitive homogeneous cell-based assay to evaluate the capacity of the expressed Nanobodies to block Herceptin® binding to HER2. The FMAT 8200 HTS system (Applied Biosystems, Foster City, Calif.) was used as described in Example 8. Bivalent 2D3-35GS-2D3 Nanobody more efficiently blocks binding of Herceptin® to HER2 as compared to monovalent 2D3 (FIGS. 15A and B). Likewise, biparatopic Nanobodies 27H3-35GS-2D3 and 27D1-35GS-2D3 block binding of Herceptin® to HER2 expressed on SKBR3 cells more efficiently than monovalent 2D3. Nanobodies 27A3, 27A5 and 30D10 have no influence on the Herceptin®-competitive characteristic of Nanobody 2D3 when fused to its N-terminal end, spaced by a 35GS linker (FIG. 15B). Finally, Nanobodies 27B7, 27C3, 27H5 and the dummy Nanobody have an inhibitory effect on the Herceptin®-competitive potential of 2D3 (FIG. 15C).

14.3 Competitive Binding of Biparatopic Nanobodies with Omnitarg-Fab to HER2.

Biparatopic Nanobodies were screened in an Omnitarg-Fab competitive homogeneous cell-based assay to evaluate the capacity of the expressed Nanobodies to block Omnitarg-Fab binding to HER2. The FMAT 8200 HTS system (Applied Biosystems, Foster City, Calif.) was used as described in Example 9. Biparatopic Nanobodies 2D3-35GS-2D3, 27H3-35GS-2D3, 27D1-35GS-2D3, 27A3-35GS-2D3, 27A5-35GS-2D3 and 30D10-35GS-2D3 did not efficiently block the binding of biotinylated Omnitarg Fab (FIG. 16). Non-labeled Omnitarg-Fab inhibited binding of biotinylated Omnitarg-Fab in a dose-dependent manner.

Example 15 Biparatopic Nanobodies Comprising a Herceptin®-Competitive and a HER2-Binding Nanobodies Inhibit SKBR3 Cell Proliferation

The growth inhibitory characteristics of biparatopic Nanobodies were evaluated using the breast tumor cell line SKBR3. Briefly, SKBR3 cells were detached using 0.25% (vol/vol) trypsin and suspended in DMEM supplemented with 10% fetal calf serum (FCS), glutamine, and penicillin-streptomycin at a density of 1×10⁵ cells/ml. Aliquots of 200 μl (2×10⁴ cells) were plated into 96-well microdilution plates and allowed to adhere. After overnight adherence, cells were washed with serum-free medium and starved for 4 hours in 100 μl serum-free medium. Then, 100 μl of 1% FCS containing medium alone or 90 μl of 1% FCS containing medium with serial dilutions of IMAC/SEC purified biparatopic Nanobodies, monovalent 2D3 or 50 nM Herceptin® was added. After 2 days of incubation, cells were pulsed with 1 μCi [³H]-thymidine and incubated for an additional 24 h prior to freezing at −80° C. Cells were subsequently thawed and embedded on glass fiber membranes using a cell harvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA). After several washings with water, filters were air-dried and counted using a γ-counter (Perkin Elmer Life Sciences).

Biparatopic Nanobodies are able to inhibit cell proliferation of SKBR3 cells to an equal or greater extent than Herceptin®. FIG. 17 shows an example of this cell proliferation assay.

Example 16 Biparatopic Nanobodies Comprising a Herceptin®-Competitive and a HER2-Binding Nanobody Inhibit AKT Signal Transduction in SKBR3 Breast Cancer Cells

Upon overexpression, HER2 may be activated by homodimerisation. HER2 plays a major regulatory role in the signalling network involved in many cellular processes, including the p21Ras/Mitogen-Activated Protein Kinase (MAPK) and PI3K/AKT pathways. Treatment of HER2 overexpressing SKBR3 cells with Herceptin® results in reduction in HER2 phosphorylation which is linked to inhibition of AKT phosphorylation.

To assess the effect of biparatopic Nanobodies on the AKT pathway in SKBR3 cells, cells were plated in 2% serum containing medium in 24-well culture plates. The next day, medium was refreshed and 50 nM of either biparatopic Nanobody. Herceptin®, monovalent 2D3 Nanobody or medium alone was added and incubated for 16 h. The reaction was stopped by aspirating the cell medium. Cells were lysed by addition of lysis buffer (20 mM NP40, 20 mM Tris-HCl pH8, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, complete protease inhibitor cocktail, 1% PBS). Protein concentration in the lysates was measured using BCA protein assay kit (Pierce) according to the manufacturer's indications. Equal amounts of protein were run on 10% polyacrylamide gels and electroblotted onto Invitrolon PVDF membranes (Invitrogen, Paisley, UK). The presence of phosphorylated AKT was assessed by probing the blots with Phospho-AKT (Ser473) antibody (Cell Signaling, Danvers, Mass.) and total AKT was detected using AKT antibody (Cell Signaling). The blots were visualized using a chemiluminescent substrate (Perkin Elmer, Wellesley, Mass., USA).

As shown in FIG. 18, biparatopic Nanobodies 27A5-35GS-2D3 and 27A3-35GS-2D3 significantly block AKT activation in SKBR3 cells, whereas dummy-2D3 biparatopic and monovalent 2D3 Nanobody do not have a visible effect on AKT signalling.

Example 17 Construction of Biparatopic Nanobodies Combining Herceptin®- and Omnitarg Competitive Nanobodies

For the construction of biparatopics consisting of a Herceptin®-competitive and Omnitarg-competitive Nanobody, the expression vector described in Example 13.1 was used. Herceptin®-competitive Nanobodies 2D3 and 5F7 were cloned either at the C-terminal or N-terminal end of Omnitarg-competitive Nanobody 47D5, spaced by a 35GS linker. Biparatopic Nanobodies 2D3-35GS-47D5, 47D5-35GS-2D3, 5F7-35GS-47D5 and 47D5-35GS-5F7 were expressed in E. coli as c-myc, His6-tagged proteins and subsequently purified from the culture medium by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Two control biparatopic Nanobody consisting of a dummy Nanobody genetically fused to the 2D3 or 47D5 Nanobody, spaced by a 35GS linker were used as controls.

Example 18 Characterization of Biparatopic Formats Combining Herceptin®- and Omnitarg Competitive Nanobodies

18.1 Biacore Analysis

A kinetic analysis for 2D3, 5F7 and 47D5 was performed on Biacore to determine the binding affinity to HER2. In addition, the influence of a dummy Nanobody fused to the N-terminal end of 2D3 and 47D5 on the binding characteristics of the latter to HER2, was analyzed. rhErbB2-Fc was immobilized on a CM5 sensor chip surface docked in T100. Approximately 3600RU of rhErbB2-Fc was immobilized. Experiments were performed at 25° C. Different concentrations of Nanobody (100 nM-0.78 nM) were made in running buffer (HBS-EP). The samples were injected for 1 min at a flow rate of 45 μl/min over the activated and reference surfaces.

In Table 2 an overview of k_(d)/k_(off), k_(a), and K_(d) values for the Nanobodies is shown. Fusion of a Nanobody at the N-terminal end of the Nanobodies 2D3 and 47D5 does not significantly alter the binding characteristics of these Nanobodies to HER2.

The binding of the biparatopic 2D3-35GS-47D5 to HER2 was compared to the binding of the monovalent building blocks 2D3 and 47D5. Hereto, approximately 90 RU of the respective Nanobodies were immobilized and different concentrations (100-1000 nM) HER2-ECD was injected. As shown in Table C-4, the off-rate of the HER2-ECD from the 2D3-47D5 surface was 25× lower than the off-rate on each of the 2D3 and 47D5 surfaces, indicating an avidity effect caused by binding of HER2-ECD on both the 2D3 and 47D5 Nanobodies simultaneously (FIG. 19).

18.2 Biparatopic Nanobodies Combining Herceptin®- and Omnitarg Competitive Nanobodies Inhibit Heregulin-Mediated HER2-HER3 Signaling

After ligand-binding, the HER receptors become activated by receptor dimerization between either two identical receptors (homodimerization) or different receptors of the same family (heterodimerization). After receptor dimerization, activation of the intrinsic protein kinase activity and tyrosine autophosphorylation occurs, recruiting and phosphorylating several intracellular substrates involving the Ras-Raf-MAPK, the PI3K/Akt, and other signaling pathways that regulate multiple biological processes including apoptosis and cellular proliferation. The mitogen-activated protein kinases (Erk1/Erk2) are one of the key endpoints in signal transduction pathways that ultimately trigger cancer cells to divide.

The ability of the biparatopic Nanobodies combining Herceptin® and Omnitarg-competitive Nanobodies to inhibit heregulin (HRG) activation of MAPK-Erk1/Erk2 was assessed in the following way. MCF7 cells (5×10⁴/well) were plated in serum-containing media in 24-well culture plates. The next day, media were removed and fresh media containing 0.1 serum were added to each well. The next day, prior to the assay, the media were replaced with serum-free medium. Cells were then incubated for 30 min with 50 nM of biparatopic Nanobody 2D3-35GS-47D5, 47D5-35GS-2D3, 5F7-35GS-47D5 or 47D5-35GS-5F7, monovalent 2D3, 5F7 or 47D5, Omnitarg-Fab or Herceptin®. Cells were then treated with 0.2 nM HRG for 15 min. The reaction was stopped by aspirating the cell medium. Cells were lysed by addition of lysis buffer (20 mM NP40, 20 mM Tris-HCl pH8, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, complete protease inhibitor cocktail, 1% PBS). Protein concentration in the lysates was measured using BCA protein assay kit (Pierce) according to the manufacturer's indications. Equal amounts of protein were run on 10% polyacrylamide gels and electroblotted onto Invitrolon PVDF membranes (Invitrogen, Paisley, UK). The presence of phosphorylated Erk1/Erk2 (p44/42 MAPK) was assessed by probing the blots with phosphor-p44/42 MAPK (Thr202/Tyr204) antibody (Cell Signaling, Danvers, Mass.) and total MAPK was detected using p44/42 MAP kinase (137F5) rabbit mAb (Cell Signaling). The blots were visualized using a chemiluminescent substrate (Perkin Elmer, Wellesley, Mass., USA).

As shown in FIG. 20, biparatopic Nanobodies 2D3-35GS-47D5 and 5F7-35GS-47D5 significantly block HRG-mediated activation of MAPK to a greater extent than Omnitarg-Fab and Herceptin®, Surprisingly, when the Omnitarg-competing Nanobody 47D5 comprised the N-terminal Nanobody in the biparatopic constructs, i.e. 47D5-35GS-2D3 and 47D5-35GS-5F7, no significant reduction in MAPK activation could be observed. Monovalent Nanobodies 2D3, 5F7 and 47D5 could not block HRG-mediated MAPK activation in MCF-7 cells.

These data suggest that the position of the Nanobodies within the biparatopic Nanobody greatly influences the potency of the molecule. In addition, the length of the linker used to genetically fuse 2 Nanobodies biparatopic may be critically important to provide maximal flexibility between the 2 Nanobodies to allow tight binding to their respective binding epitope on HER2.

Biparatopic Nanobodies 2D3-35GS-47D5 and 5F7-35GS-47D5 were also shown to inhibit heregulin (HRG)-dependent Akt activation (FIG. 21). Activation of the PI3K signal transduction pathway is important for cell survival. Complexes formed between HER2 and either HER3 or EGFR can initiate these pathways in response to HRG. Incubation of MCF7 breast cancer cells with biparatopic Nanobodies 2D3-35GS-47D5 or 5F7-35GS-47D5 inhibited HRG-mediated. Akt activation to a greater extent than Omnitarg-Fab or Herceptin®. These data suggest that the biparatopic Nanobodies 2D3-35GS-47D5 or 5F7-35GS-47D5 may inhibit HER2 ligand-activation of P13 kinase and that this inhibition may lead to apoptosis.

Example 19 In-Silico Design of Optimal Linker Lengths in Biparatopic Nanobody Formats

In-silico design of optimal linker lengths for a biparatopic Nanobody format may for example be performed as follows. The 3-dimensional (3D) coordinates of the binding mode of each individual Nanobody to its respective epitope on the target are determined, for example from:

-   -   a. a structure of the Nanobody-target complex determined by         X-ray experiments or NMR experiments.     -   b. a docking model of each Nanobody binding on their respective         epitope on the target. Also a number of potential binding modes         of each Nanobody to the target derived from docking studies can         be used. Docking can be done by e.g. ZDock (Chen and Weng 2002,         Proteins 47(3): 281-294; Chen and Weng 2003, Proteins 51(3):         397-408; Chen et al. 2003, Proteins 52(1): 80-87) and refined by         RDock (Li et al. 2003, Proteins 53(3): 693-707) or by other         methods (Fernandez-Recio et al. 2003, Proteins 52(1): 113-117).     -   c. Binding mode of each Nanobody can be extracted from the same         structure or from separate complexes. In the latter case, the         binding modes of each Nanobody on a different epitope on the         same target can be deduced by structural superposition of the         different complexes.

A linker with a given sequence and thus of given length can be modelled between the 2 Nanobodies in different ways:

-   -   a. By homology modelling (Sali, and Blundell J. 1993, Mol. Biol.         234: 779-815)         -   i. The sequence of a construct Nanobody1-linker-Nanobody2 or             Nanobody2-linker-Nanobody1 is drawn and stored in a readable             sequence format (e.g. Fasta)         -   ii. The 3-dimensional coordinates of the biparatopic             construct is built by homology modelling by using the             3-dimensional coordinates (from X-ray, NMR or docking             experiments) of the individual binding modes of the             Nanobodies as a template.     -   b. By de-novo design. A linker between the 2 Nanobodies binding         on a different epitope on the same target can be build by         de-novo design (Hu, et al. 2007, Proc. Natl. Acad. Sci. USA         104(45): 17668-17673).     -   c. Several conformations of the linker are sampled and the         lowest state energy conformations (1 or more) can be considered.

As a non-limiting example, the above was performed for a biparatopic construct comprising two Nanobodies. The modelling is shown in FIGS. 22A and 22B, which show a model of Nanobody 2D3 linked to another Nanobody docked on HER-2. Both figures show that we can dock a Nanobody to a target and predict its binding mode to the target. When doing this for several Nanobodies binding on non-overlapping epitopes on the same target, we can design a linker between the Nanobodies to create a multivalent Nanobody construct.

The 3-dimensional coordinates of the in-silico generated linker in the biparatopic construct are evaluated on at least one of the following criteria:

-   -   a. Internal energy strain of the linker; possibly compared to a         set of generated linkers of the same sequence in the free state.         At least one but preferentially several energy terms are used         (e.g. Van der Waals energy, electrostatics energy, dihedral         angle deformation energy, etc.). To calculate the energy values         an atom-based force-field (e.g. CHARMM (Brooks et al. 1983, J.         Comp. Chem. 4: 187-217)) or other means of calculating potential         energy (e.g. potentials of mean force (Muegge and Martin         1999, J. Med. Chem. 42: 791)) can be used.     -   b. Internal energy strain on at least one of the residues         (amino-acids) of the linker. For three biparatopic constructs         5F7-35GS-47D5, 47D5-35GS-5F7 and 47D5-40GS-5F7 energy penalty         values were calculated for each residue in the linker as well as         for 10 residues of each Nanobody connected to the linker. Energy         values are shown in FIGS. 23, 24 and 25.     -   c. The root-mean square deviation (RMSD) between the         3-dimensional coordinates of the 2 Nanobodies in the biparatopic         construct and the 2 Nanobodies in their non-linked (monovalent)         binding mode. The higher this value the less likely this linker         is appropriate. FIG. 26 shows the backbone RMSD (Å²) between the         5F7-linker-47D5 constructs (built by homology modelling) with         the individual Nanobodies 5F7 and 47D5 in their unlinked binding         mode. The linker length varies from 5 to 35. FIG. 26A shows that         the RMSD-value is at a minimum value with linker lengths larger         or equal to 15 residues. When shorter linkers are used (e.g.         linker length=5, 10) we see an increased RMSD indicating that         the Nanobodies in the bivalent construct are deviating from         their monovalent binding mode. These in-silico experiments         suggest that biparatopic constructs with linker lengths lower         than 15 residues will have a significant deviation from the         optimal binding mode of the individual Nanobodies to the target.         In FIG. 26B a ribbon view is shown of the 5F7-linker-47D5         biparatopic construct for 2 linker lengths. The binding mode of         the individual Nanobodies and the biparatopic constructs are         shown. When a 35GS linker is used between the 2 Nanobodies and a         very limited deviation from the individual binding modes is         observed. However, when a 5GS linker is used, both Nanobodies in         the biparatopic construct significantly deviate from their         optimal binding mode.     -   d. Scores from scoring functions in homology modelling protocols         which are derived based on a combination of experimental data         and in-silico results (Sali & Overington, Protein Science         3(9):1582-1596, 1994).

As can be seen from the above results, the linker in this specific example should preferably be at least 15 amino acids in length, with linkers of between 20 and 40 amino acid residues, such as about 25, 30 or 35 amino acid residues, being particularly suited.

Also, constructs with different potentially suitable linker lengths (as determined by the above in silico analysis) may be prepared and tested for affinity/avidity, specificity, or potency using suitable binding assays or in vitro or in vivo potency assays, for example those mentioned in the present specification. In this way, optimal linker length may be determined, confirmed or verified.

Example 20 Construction of Multiparatopic Nanobodies for Broader Biological Activity

Simultaneous binding of 2 adjacent, non-overlapping epitopes by both arms of a biparatopic Nanobody without significant loss of entropy endows biparatopic Nanobodies with increased binding affinity to the target and as a result, higher potency can be obtained. The engineering of Nanobody fragments to obtain an increased potency or broader activity is not limited to the construction of biparatopic Nanobody fragments. Engineering of triparatopic and even tetratopic Nanobodies with careful selection of the epitopes targeted on the antigen, combined with rational design of linkers to allow maximal flexibility of the binding domains within the multiparatopic antibody, may for example result in the blocking of several critical interaction sites of the target, leading to improved potency and even an unparalleled biological activity.

TABLES

TABLE B-1 Preferred Nanobodies against HER2 obtained as described in Example 3 <Name, SEQ ID #; PRT (protein); -> <13D11, SEQ ID NO: 2051; PRT; -> EVQLVESGGGLVHPGGSLRLSCVGSGFSLDDYGMTW VRRAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCGQGWKIVPTNPRGHGTQVTVSS <2B4, SEQ ID NO: 2052; PRT; -> EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS <2G2, SEQ ID NO: 2053; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYTDPVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNRGWKIVPTDLGGHGTQVTVSS <13D2, SEQ ID NO: 2054; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLRSEDTAVYSCNQGWKIVPTDRGGHGTQVTVSS <2D5, SEQ ID NO: 2055; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS <2F4, SEQ ID NO: 2056; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRRGHGTQVTVSS <2C3, SEQ ID NO: 2057; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS <17E3, SEQ ID NO: 2058; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGW YRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLTPEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS <17H3, SEQ ID NO: 2059; PRT; -> EVQLMESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS <17D2, SEQ ID NO: 2060; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGSHGTQVTVSS <2F1, SEQ ID NO: 2061; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKELEWISSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIVPMDRRGHGTQVTVSS <2E2, SEQ ID NO: 2062; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS <2C2, SEQ ID NO: 2063; PRT; -> EVQLVESGGGLVQGGSLRLSCVASGFSLDDYAMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNARNTLFLQMNSLTPEDTAIYYCNQGWKILPTDRRGHGTQVTVSS <2E3, SEQ ID NO: 2064; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS <13B10, SEQ ID NO: 2065; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGFEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS <2D1, SEQ ID NO: 2066; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNRGWKILPTNRGSHGTQVTVSS <2H3, SEQ ID NO: 2067; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS <2H1, SEQ ID NO: 2068; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVRGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS <2C1, SEQ ID NO: 2069; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS <15C5, SEQ ID NO: 2070; PRT; -> EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWNVTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS <2B3, SEQ ID NO: 2071; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDCADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS <29H2, SEQ ID NO: 2072; PRT; -> EVQLVESGGGLVQGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS <17E4, SEQ ID NO: 2073; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS <17A2, SEQ ID NO: 2074; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNKGWKVWPTDRGTHGTQVTVSS <15D1, SEQ ID NO: 2075; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKVWPTDRGTHGTQVTVSS <17B8, SEQ ID NO: 2076; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS <15C11, SEQ ID NO: 2077; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTPVTVSS <15G8, SEQ ID NO: 2078; PRT; -> EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWNGTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPENTAVYYCNQGWKILPAERRGHGTVTVSS <17H4, SEQ ID NO: 2079; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLINYAMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLHMNNLSPEDTAVYYCGQGWKIHPADRGGHGTQVTVSS <27G8, SEQ ID NO: 2080; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS <38C6, SEQ ID NO: 2081; PRT; -> EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTW VRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQWKIRPTIPMGHGTQVTVSS <2A4, SEQ ID NO: 2082; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSW VRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS <15G7, SEQ ID NO: 2083; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSW VRQAPGKGLEWVSAINWSGTHRNYADSVKGRFTISRDNNKKTVYLQMNSLKSEDTAVYYCATGWQSTTKNQGYWGQGTQVTVSS <15B7, SEQ ID NO: 2084; PRT; -> EVQLVESGGGLVQPGGSLKLSCAASGFIFDDYAMSW VRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKSQGYWGQGTQVTVSS <5G4, SEQ ID NO: 2085; PRT; -> EVQLVESGGGLVQPGGSLTLSCAGSGFIFDDYAMSW VRQAPGKGLEWVSSINWSGSHRNYADSVKGRFTISRDNAKKTLYLQMNSLKSEDTAVYYCATGWQSTTKNQNYWGQGTQVTVSS <13B2, SEQ ID NO: 2086; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEW ISSINWSGTHKDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <2E5, SEQ ID NO: 2087; PRT; -> EVQLVESGGSLVQPGESLRLSCAASGFTFDDYAMSWVRQAPGKGLEW ISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <15G1, SEQ ID NO: 2088; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27B1, SEQ ID NO: 2089; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEW ISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <17E7, SEQ ID NO: 2090; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEW VSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <17D8, SEQ ID NO: 2091; PRT; -> EVQLVESGGSLVPPGGSLRLSCAVSGFTFDDYAMSWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNMLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <5F8, SEQ ID NO: 2092; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYALSWVRQAPGKGLEW ISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <2D4, SEQ ID NO: 2093; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS <13D8, SEQ ID NO: 2094; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <17G8, SEQ ID NO: 2095; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEW VSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <2H4, SEQ ID NO: 2096; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <2F3, SEQ ID NO: 2097; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYTGSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS <2F5, SEQ ID NO: 2098; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLOMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <30E10, SEQ ID NO: 2099; PRT; -> KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <29H1, SEQ ID NO: 2100; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEW VSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <17E2, SEQ ID NO: 2101; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <2B1, SEQ ID NO: 2102; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS <2A5, SEQ ID NO: 2103; PRT; -> EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <13C12, SEQ ID NO: 2104; PRT; -> EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQTVSS <17E10, SEQ ID NO: 2105; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDCTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <27D4, SEQ ID NO: 2106; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEW VSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQTVSS <15F9, SEQ ID NO: 2107; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYTGSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <30H9, SEQ ID NO: 2108; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <39C1, SEQ ID NO: 2109; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS <27G2, SEQ ID NO: 2110; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQTPGKGLEW VSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS <2D3, SEQ ID NO: 2111; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEW VSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <5F7, SEQ ID NO: 2112; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQREL VALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS <47D5, SEQ ID NO: 2114; PRT; -> KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQREL VALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS <14B11, SEQ ID NO: 2115; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYGMGWFRQVPGKEREF VATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTARYYCGVETYGSGSSLMTEYDYWGQGTQVTVSS <14B10, SEQ ID NO: 2116; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREF VATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS <14B4, SEQ ID NO: 2117; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREF VATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPEDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS <14C11, SEQ ID NO: 2118; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREF VATINWSGATAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS <14B5, SEQ ID NO: 2119; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREF VATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETFGSGSSLMSEYDYWGQGTQVTVSS <14C6, SEQ ID NO: 2120; PRT; -> EVQLVESGGGSVQAGGSLRLSCVASEGTFSSYGMGWFRQAPGKERAF VATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATDTYGSGSSLMNEYDYWGQGTQVTVSS <14A4, SEQ ID NO: 2121; PRT; -> EVQLVESGGGSVQAGSSLTLSCVASEGTFSSYGMGWFRQAPGKERAF VATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS <14B3, SEQ ID NO: 2122; PRT; -> EVQLVESGGGLVQPGGSLTLSCVASEGTFSSYGMGWFRQAPGKERAF VATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS <14C1, SEQ ID NO: 2123; PRT; -> EVQLVESGGGSVQAGGSLRLSCAASGSTFSSYGMGWFRQAPGKERAF VATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATETYGSGSSLMNEYDYWGQGTQVTVSS <14A12, SEQ ID NO: 2124; PRT; -> EVQLVKSGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREF VATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS <14A2, SEQ ID NO: 2125; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREF VATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS <14A1, SEQ ID NO: 2126; PRT; -> EVQLVESGGGSVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREF VATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDYWGHGTQVTVSS <17C3, SEQ ID NO: 2127; PRT; -> EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREW VAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS <46D3, SEQ ID NO: 2128; PRT; -> KVQLVESGGGLVQAGGSLRLSCAASGRTFTEYSMGWFRQAPGKEREF VATISWNYGYTYYSDSVKGRFTVSRDIAENTVYLQMNTLKSEDTAVYYCAAKIGWLSIRGDEYEYWGQGTQVTVSS <27H5, SEQ ID NO: 2129; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGIGWFRQASGKEREG VSCITSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAALPFVCPSGSYSDYGDEYDYWGQGTOVTVSS <17C2, SEQ ID NO: 2130; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMSWVRQAPGKGLEW VSAVDSGGGRTDYAHSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCTKHVSDSDYTEYDYWGQGTQVTVSS <17D11, SEQ ID NO: 2131; PRT; -> EVQLVESGGGLVQAGGSLRLSCTASGRTSSTSAMGWFRQAPGKEREF VATISRGGSATYYADSLKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAARRSSLYTSSNVFEYDYWGQGTQVTVSS <15A6, SEQ ID NO: 2132; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDW VATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS <17B6, SEQ ID NO: 2133; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTMAWYRQAPGKQRDW VATIGTSGPPRYADSVKGRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADYWGQGTQVTVSS <17C5, SEQ ID NO: 2134; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDW VATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTPVTVSS <15E11, SEQ ID NO: 2135; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASRIPFSSRTMAWYRQAPGKQRDW VATISARGMPAYEDSVKGRFTVSRDNDKNTLYLQMNSLKPEDTAVYYCRDVNADYWGQGTQVTVSS <15C2, SEQ ID NO: 2136; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAQGKQRDW VATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS <2A3, SEQ ID NO: 2137; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQAPGKPRDW VATIRNGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTATYLCRDVNGDIWGQGTQVTVSS <27A5, SEQ ID NO: 2138; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDW VATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSS <2C5, SEQ ID NO: 2139; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQTPGKSRDW VATIRSGTPVYADSVKGRFTVSRDNAKNTLYLRMNSLKSEDSATYTCRAVNADIWGQGTQVTVSS <27G5, SEQ ID NO: 2140; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDW LATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS <13A9, SEQ ID NO: 2141; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASRIPASIRTMAWYRQAPGKQRDW VATIGTGGTPAYADSFKGRFTVSRDNANHTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS <29E9, SEQ ID NO: 2142; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDW LATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS <15D8, SEQ ID NO: 2143; PRT; -> EVQLVESGGGLVQPGGSLKLSCVASTIPASIRTMAWYRQTPGNQRDW LATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS <15G4, SEQ ID NO: 2144; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPFRSRTMAWYRQAPGKTRDW VATIGTHGTPLYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCWDVNGDYWGQGTQVTVSS <15D12, SEQ ID NO: 2145; PRT; -> EVQLVESGGGLVQAGESLRLSCATSGITFKRYVMGWYRQGPGKQREL VATVNDGGTTSYADSVKGRFAISRDNAKNTAYLQMNSLKAEDTAVYYCNAVWKLPRFVDNDYWGQGTQVTVSS <15E12, SEQ ID NO: 2146; PRT; -> EVQLMESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREW VAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS <13D7, SEQ ID NO: 2147; PRT; -> EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREW VAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS <13A8, SEQ ID NO: 2148; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQR DWVATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS <15A4, SEQ ID NO: 2149; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQR DWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQINSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS <17F7, SEQ ID NO: 2150; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIAQSIRVMAWYRQPPGKQRDW VGTISSDGTANYADSVKGRFTISRDNAKKTMYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS <15C8, SEQ ID NO: 2151; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDW VATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS <17A10, SEQ ID NO: 2152; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPSIRAIAWYRQAPGKQRDW VATSGTGYGATYDDSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS <27D3, SEQ ID NO: 2153; PRT; -> EVQLMESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDW VATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS <13B12, SEQ ID NO: 2154; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDW VATIGSDGTTIYADSVKGRFTLSRHNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS <15B2, SEQ ID NO: 2155; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDW VATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS <15B11, SEQ ID NO: 2156; PRT; -> EVQLVESGGGSVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDW VATIYSRSGGAVYADSVKGRFTISSDNAKNTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS <13C9, SEQ ID NO: 2157; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPSIHAMAWYRQAPGKQRDW GATTYSRGGTTYNDSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS <17D5, SEQ ID NO: 2158; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGIIGTIRTMAWYRQAPGKQRDW VASIGTRGAPVYADSVNGRFTISRDGATNTVFLQMNNLKPEDTAVYYCRDVNRDYWGQGTQVTVSS <27B5, SEQ ID NO: 2159; PRT; -> EVQLVESGGGLVQAGGSLRLPCAASGIAFRIRTMAWYRQAPGKQRDW VATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS <27C7, SEQ ID NO: 2160; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDW VATSDSGGTTLYADSVKGRFTVSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS <13D4, SEQ ID NO: 2161; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDW VATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLEPDDTAVYYCRDVNREYWGQGTQVTVSS <15G5, SEQ ID NO: 2162; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSTIRAMAWYRQAPGRQRDW VATIYSPSGSAVYADSVKGRFTISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS <13C4, SEQ ID NO: 2163; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDW VATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS <46G1, SEQ ID NO: 2164; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSDDAMGWFRQAPGKEREC VASLYLNGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSQYNYWGQGTQVTVSS <46E4, SEQ ID NO: 2165; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRAFKDDAVGWFRQAPGKEREC VASMYLDGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSEYNYWGQGTQVTVSS <17B5, SEQ ID NO: 2166; PRT; -> EVQLVESGGGLVQTGGSLRLSCAASGSTFRTDMMGWYRQAPGKQREF VASITKFGSTNYADSVKGRFTISNDNAKDTVYLQMNSLKSEDTAVYYCRNFNRDLWGQGTQVTVSS <15C9, SEQ ID NO: 2167; PRT; -> EVQLVESGGGLVQAGGSLKLSCVNSGIPSTLRAMAWYRQAPGRQRDW VATSSNTGGTTYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCRDVNRDLWGQGTQVTVSS <13D10, SEQ ID NO: 2168; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASSVITLDSNAIGWFRQAPGKERE EVSCIASSDGSTYYAESVKGRFTISKDYTRNTVYLQVNSLKPEDTAVYHCATDANPNCGLNVWNSWGQGTQVTVSS <17C6, SEQ ID NO: 2169; PRT; -> EVQLVESGGGLVQAGGSLTLSCAASGSTSSLDIMAWYRQAPEKQREL VASVSGGGNSDYASSVKGRFTISGDTAKSTLYLQMNSLKPEDTAMYYCYGRDYYYMPFWGQGTQVTVSS <15A2, SEQ ID NO: 2170; PRT; -> EVQLVESGGGLAQAGGSLSLSCAASGRFFSTRVMAWYRQTPGKQREF VASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS <17A8, SEQ ID NO: 2171; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASGRFFSTRVMAWYRQTPGKQREF VASMRGSGSTNYADSVRGRFAISRDNAKNMVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS <15G10, SEQ ID NO: 2172; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASGRFFSTRVMAWYRQTPGKQREF VASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS <27A3, SEQ ID NO: 2173; PRT; -> EVQLVESGGGLVQAGGSLSLSCVASGRFFSTRVMAWYRQTPGKQREF VASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS <17H10, SEQ ID NO: 2174; PRT; -> EVQLVESGGGLVQAGGSLSLSCSASGRFFSTRVMAWYRQTPGNQREF VATIHSSGSTIYADSVRGRFAISRDNAKNTVYLQMRSLKPEDTAVYYCRDINADQWGQGTQVTVSS <30D10, SEQ ID NO: 2175; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREW VATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS <15H4, SEQ ID NO: 2176; PRT; -> EVQLVESGGGLVQAGGSLTLSCAPSESTVSFNTVAWYRQAPGEQREW VATISRQGMSTYPDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCRDINHDIWGRGSQVTVSS <17B7, SEQ ID NO: 2177; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTMAWYRQAPGKQRDW VATIGSDGLANYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYFCRDINRDYWGQGTQVTVSS <15D2, SEQ ID NO: 2178; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGVFGPIRAMAWYRQAPGKQRDW VATIGSSGHPVYTDSVKGRFTFSKDGAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS <17G5, SEQ ID NO: 2179; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGIGIAFSSRTMAWYRQAPGKQRDW VATIGSGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS <15B6, SEQ ID NO: 2180; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGIIGSFRTMAWYRQAPGNQRDWVA TIGSAGLASYADSVRGRFTLSRDNAKKTVYLQMNSLKPEDTAIYYCRDINGDYWGQGTQVTVSS <27F2, SEQ ID NO: 2181; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTLAWYRQAPGKQRDWVA TISSAGGTAYADAVKGRFTISISRDNVEYTVDLQMDSLKPEDTAVYYCRDINGDYWGQGTQVTVSS <17F5, SEQ ID NO: 2182; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVA TIAGDGSTVYADSMKGRFTISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSS <17B2, SEQ ID NO: 2183; PRT; -> EVQLVESGGGLVQPGGSLRLSCAGSGFTFSNYAMTWVRQAPGKGLEW VSGVGGDGVGSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCTKDISTFGWGPFDYWGQGTQVTVSS <27H4, SEQ ID NO: 2184; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDL VASIDASGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS <13A4, SEQ ID NO: 2185; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDL VASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS <2A1, SEQ ID NO: 2186; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKITFRRYIMDWYRQAPGKQREL VASINSDGSTGYTDSVKGRFTISRDNTKNTLDLQMNSLKPEDTAVYYCHGRWLEIGAEYWGQGTQVTVSS <15E10, SEQ ID NO: 2187; PRT; -> EVQLVESGGGLVQAGGSLKLSCVASGITFFRYTMGWYRQAPGKEREL VAEISSADEPSFADAVKGRFTISRDNAKNTVVLQMNGLKPEDTAVYYCKGSWSYPGLTYWGKGTLVTVSS <27E7, SEQ ID NO: 2188; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKEREL VATILSEGDTNYVDPVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSS <47E5, SEQ ID NO: 2189; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASASIFGFDSMGWYRQAPGNERIL VAIISNGGTTSYRDSVKGRFTIARDNAKNTVSLQMNSLKPEDTAVYYCNLDRRSYNGRQYWGQGTQVTVSS <2G4, SEQ ID NO: 2190; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGNIFSHNAMGWYRQAPGKQREL VTYITINGIANYVDSVKGRFTISRDNTKNTMYLQMVSLKPEDTAVYYCNVGGREYSGVYYYREYWGQGTQVTVSS <14D4, SEQ ID NO: 2191; PRT; -> EVQLVESGGGLVQAGDSLRLSCAASGRALDTYVMGWFRQAPGDGREF VAHIFRSGITSYASSVKGRFTISRDNAKNTVYLQMASLKPEDTAAYYCAARPSDTTWSESSASWGQGTQVTVSS <17A5, SEQ ID NO: 2192; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYSMSWVRQATGKGLEW VSGISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLKSEDTAVYYCAKDLGNSGRGPYTNWGQGTQVTVSS <15D10, SEQ ID NO: 2193; PRT; -> EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYRMYWVRQAPGKGLEW VSAIKPDGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCGVPGFGWTFSSWGQGTQVTVSS <13C2, SEQ ID NO: 2194; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQREL VAAVDNDDNTEYSDSVAGRFTISRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS <17G11, SEQ ID NO: 2195; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRWGWYRQAPGKQREL VAAIDDGGNTEYSDFVNGRFTISRDNPETAVHLQMNSLKLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS <17A3, SEQ ID NO: 2196; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASATLHRFDNNWYRQAPGKQREL VATIAHDGSTNYANSVKGRFTISRDNARDTLFLQMHALQPEDTAVYMCNLHRWGLNYWGQGTQVTVSS <27B7, SEQ ID NO: 2197; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSAISSGGGSITTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS <17A6, SEQ ID NO: 2198; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSAISSGGGSITTYADSVKGRFTISTDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS <17D7, SEQ ID NO: 2199; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTLDYCAIGWFRQAPGKEREG VSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGSGTCYADFGSWGQGTQVTVSS <46D4, SEQ ID NO: 2200; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEW VSSINWSGTHTDYAEDMKGRFTISRDNAKKTLYLQMNSLQSEDTAVYYCAKGWGPAVTSIPVATLGTQVTVSS <27B3, SEQ ID NO: 2201; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREW VATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS <27E5, SEQ ID NO: 2202; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREW VATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS <27D6, SEQ ID NO: 2203; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREW VATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS <30D10, SEQ ID NO: 2204; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREW VATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS <47G11, SEQ ID NO: 2205; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGRIFYPMGWFRQAPGKEREFVA AIGSGDIITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSRDYSRSRDPTSYDRWGQGTQVTVSS <27C3, SEQ ID NO: 2206; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEW VSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSS

TABLE B-2 Preferred Nanobodies against HER2 obtained as described in Example 4 <Name, SEQ ID #; PRT (protein); -> <11A101/1-120, SEQ ID NO: 2207; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFNAMGWFRQAPGKEREFVAAISRS PGVTYYADSVKGRFTTSRDNAKNTVYLQMNDLKPEDTAVYYCAADFYLATLAHEYDYWGQGTQVTVSS <11A22/1-122, SEQ ID NO: 2208; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGTEREFIAGIRW SDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADFYVSTLAHEYDYWGQGTQVTVSS <12D44/1-122, SEQ ID NO: 2209; PRT; -> KVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGTEREFIAGIRW SDGSTYYADSVKGRFTISRANAKNTVYLQMNGLKPEDTAVYYCAADFYVSTLAHEYDYWGQSTQVTVSS <12E11/1-122, SEQ ID NO: 2210; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVGGIRW SDGSTYYADSVKGRFTISRDNAKITVYLQMNSLKPEDTAVYYCAADFYVSTLAHEYDYWGQGTQVTVSS <13G111/1-123, SEQ ID NO: 2211; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERAFVAAIRW SGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTFTLSTLSHEYDYWGQGTQVTVSS <13F71/1-123, SEQ ID NO: 2212; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGRTFSNYALAWFRQAPGKEREFVAAINW RSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLIVATLPGEYDYWGQGTQVTVSS <14H61/1-122, SEQ ID NO: 2213; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSRFAMGWFRQAPGKEREFVAAVRW SDDYTYYADSVKGRFTISRDNAKNTVYLQMNSLSPEDTAVYYCAADEILATLPHEYDYWGQGTQVTVSS <22B12/1-124, SEQ ID NO: 2214; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVAGINK SGGITHSADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADAYTVIATLPHEYDYWGQGTQTVSS <14H71/1-123, SEQ ID NO: 2215; PRT; -> EVQLVESGGGLVQAGGSLRLSCEASGLTISSLTMAWFRQAPGKEREFVANIK WSGDRIVYADSVKGRFTISRDSAKNAVNLQMELVESDDTAVYYCAAKHSTVAGLTHEYDYWGQGTQVTVSS <12D51/1-120, SEQ ID NO: 2216; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGSAFSIKSMGWYRQAPGKQRELAAVII SSGTTTYADSVKGRFTISRDSAKNTVYLQMDSLKPEDTAVYVCNAVYVSTWGNGYDYWGQGTQVTVSS <11A111/1-126, SEQ ID NO: 2217; PRT; -> EVQLVESGGGLVQAGGSLGLSCAAAGRTFSSSLMGWFRQAPGKEREFVAAIT DNGGSTYYADSVKGRFTISRDNAKNSVYLQMNSLKPEDTAIYYCAARRSGYYSLSTSPHQYAYWGQGTQVTVSS <13G71/1-124, SEQ ID NO: 2218; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAMGWFRQAPGKERDFVAAIT SSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCGARVNYAAYSRLEHDYHYWGQGTQVTVSS <13G74/1-125, SEQ ID NO: 2219; PRT; -> EVQLVESGGGLVQAGGSLRLSCATSGRTFSTYASMGWFRQTPGKEREFVAAI TSSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCGARVNYAAYSRLEHDYHYWGQGTQVTVSS <11A71A/1-116, SEQ ID NO: 2220; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGNIDGIITMGWYRQRPGKPREWVGTIN SGGDTNYAGSVKGRFTIARDDAKNTMYLQMNGMKPEDTAVYYCKMNRAGIYEYWGQGTQVTVSS <22B101/1-123, SEQ ID NO: 2221; PRT; -> EVQLVESGGGLVQTGGSLRLSCAASGPTFSDYAIGWFRQAPGKEREFVAAIS SSGISTIYGDSVKGRFDISRDNAKNTVYLQMNRLKPEDTAVYYCAARLFMATPNQGQYYYWGQGTQTVSS <11B42/1-123, SEQ ID NO: 2222; PRT; -> EVQLVESGGGLVQAGDSLRLSCAARSGFTFSNHIMGWFRQAPGKERELIAEI TWNGGSTYYADSVKGRFAISRDNALNTVYLQMNSLKPEDTAVYYCAARPSYSTNNVKSYRYWGQGTQVTVSS <13E111/1-124, SEQ ID NO: 2223; PRT; -> EVQLVESGGGLVQAGSSLRLSCALSGRTFSDYAIGWFRQAPGKEREFVAAIS GWSGGTTNYADSVKGRFTISRDNGKNTVDLRMNSLKPEDTAVYYCAARPAVVHTRKESYPYWGQGTQVTVSS <14H12/1-125, SEQ ID NO: 2224; PRT; -> EVQLVESGGGLVQAGGSLRLSCIASERTFSSAGVGWFRQAPGKERDFVAAIS WNGVTIYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARINYSVLTTTSSSYHYWGQGTQVTVSS <13G101/1-123, SEQ ID NO: 2225; PRT; -> EVQLVESGGGLVQPGDSLRLSCSASEGTLSRSRVAWFRQAPGKEREFVTVI SGVGTSYADSVKGRFTISRDDAKNTVYLQMNSLKAEDTAIYYCAADFRSTWLSSSGSSYTYWGQGTQVTVSS <13G41/1-121, SEQ ID NO: 2226; PRT; -> EVQLVESGGGLVQPGGSLTLSCVGSGRRFSADVMGWYRQAPGKQREFVASI SSGSAINYADSVKGRFTVSRDNAQNTVYLQMNSLKIEDTGVYYCNARRIVNVEGAYRDYWGQGTQVTVSS <22B910/1-121, SEQ ID NO: 2227; PRT; -> EVQLVESGGGLVQPGGSLPLSCAASGSIFRMNDMGWYRQAPGKQRERVATL TSAGNTNYADSVKGRFTISGDDARNTVYLQMNSLNPEDTAVYYCNAKVVVAVEGAKYDYWGQGTQVTVSS <21A81/1-122, SEQ ID NO: 2228; PRT; -> EVQLVESGGGLAQAGGSLRLSCAVFGRSRYGMAWFRRAPGKEREFVAGIAW NGASIGSADSVRGRFTISRDNSENTVYFEMGSLKPEDTAVYYCAICRISWCAGAESDYGYWGQGTQVTVSS <21A92/1-127, SEQ ID NO: 2229; PRT; -> EVQLVESGGGQVQAGGSLRLSCTESGRAFNTRAMGWFRQAPEKEREFVAGI TMSGFNTRYADSVKGRFTISRDNAKGTVYLQMSSLKPEDTAVYYCAADSITDRRSVAVAHTSYYYWGQGTQVTVSS <22C712/1-123, SEQ ID NO: 2230; PRT; -> EVQLVESGGGLVQAGGSLGLSCAASGRTFSNYAMGWFRQAPGKEREFVAGI SWSGGHTFYADSVKGRFTISRDNTKNTVYLQMNSMRPEDTAVYYCAARLSSVAVASTRYDYWGQGTQVTVSS <11A13/1-125, SEQ ID NO: 2231; PRT; -> EVQLVESGGGLVQAGDSLRLSCVASGGTFGSYAMGWFRQAPGKEREFVATI DWSGDTAFYADSVKGRFTISRDIANDVVYLQMNSLEPEDTAVYYCARNRQSGVASENLRLYTYWGQGTQVTVSS <13G93/1-123, SEQ ID NO: 2232; PRT; -> EVQLVESGGGLAQAGDSLRLSCVDSGSSFSAYAMGWFRQAPGKEREFVAAV SWDGRNTYYADSVKGRFTISRDNAKNTLYLQTTSLRPEDTGVYYCAEDKQSGVSVNPKYAYWGQGTQVTVSS <12C52/1-118, SEQ ID NO: 2233; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSGGTFESDTMAWFRQAPGKEREFVARV SWIRTTYYSDSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYCAAQTLGRSLYDYWGQGTQVTVSS <12C61/1-126, SEQ ID NO: 2234; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSNAMAWFRQAPGNERELVSAI GWSGASTYYIDSVEGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASRYSGGVATARRSEYHYWGQGTQVTVSS <21A61/1-125, SEQ ID NO: 2235; PRT; -> EVQLVESGGGLVQAGDSLRLSCVASGDSFNTYTMGWFRQAPGKEREFVAAI RWSGGTTFYGDSVKGRFTISRDYAKNTWYLQMNTLKPEDTAAYYCAAVATYSRNVGSVRNYDYWGQGTQVTVSS <11A121/1-126, SEQ ID NO: 2236; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSEGTFSSYSMGWFRQAPGKDREFVSAI TWNGTRTYYRDSVKGRFTISRDNAKNTVQLQMNSLKPEDTAVYYCAVSQPLNYYTYYDARRYDYWGQGTQVTVSS <11A91/1-124, SEQ ID NO: 2237; PRT; -> EAQLVESGGGLVQAGGSLRLSCTASGRTYSTIMGWFRQAPGKEREFVAAIR WSGGSAFYADSVKGRFTISRDNAKNTVYLQMTSLMPEDTAVYYCADTPVYYQRYYDQNAYDYWGQGTQVTVSS <13G72/1-118, SEQ ID NO: 2238; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAMGWFRQAPGKERDFVAAI TSSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKYYSYYAYDYWGQGTQVTVSS <13E81/1-124, SEQ ID NO: 2239; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFSVYHMAWFRQAPGKEREFVAAI RSSGGLFYALSVSGRFTISRDNAKDTMYLQMNVLKPEDTAVYYCAASPVYYIDYSSQYKYGYWGQGTQVTVSS <11B31/1-124, SEQ ID NO: 2240; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGAFGVYHMGWFRQAPGKEREFVAAI RSGGTTLYEDSVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYYCATQIYYRTNYYSQNAYDYWGQGTQVTVSS <13G81/1-124, SEQ ID NO: 2241; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYHMGWFRQAPGKEREFVAVI RSGGTTLYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYLCAAQIYYRTNYYSQNNYDYWGQGTQVTVSS <21A53/1-124, SEQ ID NO: 2242; PRT; -> EVQLVESGGGLVQAGGSLELSCAASGGAFGVYHMGWFRQAPGKEREFVAAI RSGGTTLYEDSVKGRVTISRDDAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYYSQNVYDYWGQGTQVTVSS <14H51/1-124, SEQ ID NO: 2243; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYTMAWFRQAPGKEREFVAAI RSGATTLYEDSVKGRFTISRDDAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYYSQNEYDYWGQGTQVTVSS <21A21/1-124, SEQ ID NO: 2244; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYHMGWFRQAPGTEREFVAVI RSGGTTLYEDSVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYSSQSNYDYWGQGTQVTVSS <21A111/1-124, SEQ ID NO: 2245; PRT; -> EVQLVESGGGLVQAGGSLKLSCAVSGRTIVPYTMAWFRQAPGKEREFVAV TRSGGTTFYADSAKGRFTIARDDAKNTVYLQMNSLKPEDTAVYYCALATAYRTNYSSRDKYDYWGQGTQVTVSS <22B1212/1-122, SEQ ID NO: 2246; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI NSGGGSTSYADSVNGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKYLSFYSDYEVYDYWGQGTQVTVSS <11A31/1-120, SEQ ID NO: 2247; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFSSGVMAWFRQSPGEEREFLALI TRNGETKKTADSVKGRFTISRDNAKNGVSLQMDSLKAEDTAVYYCASDPTYGSGRWTYWGQGTQVTVSS <13E51/1-128, SEQ ID NO: 2248; PRT; -> EVQLVESGGGLNQAGGSLRLSCAASRHTFSGYAMGWFRQAPGKEREFVAAI RWSGGITYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTALYYCARSVTYYSGSHAYTQEGGYARWGQGTQVTVSS <12D121/1-126, SEQ ID NO: 2249; PRT; -> EVQLVESGGGLVQTGGSLRLSCAASGRAFSTYGMGWFRQAPGKAREFVAAI SRSGTGTYYAGSMKGRFTISRDDAKNTVYLOMNSLKPEDTAVYYCAARQPYASGSHYSSTQYTYWGQGTQVTVSS <13F121/1-119, SEQ ID NO: 2250; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRSFNDYTMGWFRQTPGKEREFVARV WWNGGSAYYADSVKGRFTISIDNAKNTVYLQMNNLTPEDTAVYYCAALYRGRSVYDDWGQGTQVTVSS <13G121/1-127, SEQ ID NO: 2251; PRT; -> EVQLVESGGGLVRAGTSLRLSCADSARTFSSAAMGWFRQAPGKEREFVSAI SPIGSSKYYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAASSYGSTYYSQGRAYYYDYWGQGTGVTVSS <22B41/1-124, SEQ ID NO: 2252; PRT; -> EVQLVESGGGLVQPGGSLRLSCTVFGRTFSGDVIGWFRQAPGKEREFVAAI STSGGGTDSADSVKGRFTISKENAKNTVYLQMTILKPEDTAVYYCASSPYGPLYRSTHYYDYWGQGTQVTVSS <12D71/1-125, SEQ ID NO: 2253; PRT; -> EVQLVESGGGLVQAGGSLGLSCAASGRTVSTMGWFRQAPGKEREFVTAITW SGDSTNFADSVKGRFTISRDSAKDTVYLQMNNLKPEDTAVYYCAATTYYSGSYISTLSTSYNYWGQGTQVPVSS <13F42/1-111, SEQ ID NO: 2254; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGVGWFRQAPGKGRESVATI FVGGTTYYSDSVKGRFTISRDNAKNAVNLQMSNLKPEDTALHYCTICSYRGQGTQVTVSS <12C101/1-111, SEQ ID NO: 2255; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGVGWFRQAPGKERESVATI FVGGTTYYSDSVKGRFTISRDNARNAVNPQMNNLKPEDTAVYYCTIGSYRGQGTQVTVSS <14H91/1-127, SEQ ID NO: 2256; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSRDVMGWFRQAPGKEREFVAAK TWSGASTYYADSVRGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCAARDSSTLDSTYYVGGSYNYWGRGTQVTVSS <13F41/1-111, SEQ ID NO: 2257; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGVGWFRQAPGKERESVATI FVGGTTYYSDSVKGRFTISRDNAKNAVNLQMSNLKPEDTALYYCTIGSYRGQGTQVTVSS <14H21/1-125, SEQ ID NO: 2258; PRT; -> EVQLVESGGGLVQGGSLRLSCVRSGGYFGSYHIGWFRQAPGNEREFVAAI TWNGASTSYADSVKGRFTISRSIAENTVYLQMNKVKPEDTAVYYCAARMYGSDWLPRPEDFDSWGQGTQVTVSS <22B610/1-120, SEQ ID NO: 2259; PRT; -> EVQLVESOGGLVQPGGSLRLSCAASGSIFSINAMGWYRPAPOKQRELVARI TSTGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADVSPSYGSRWYGWGQGTQVTVSS <12C32/1-127, SEQ ID NO: 2260; PRT; -> EMQLVESGGGLVQAGGSLRLSCATSERTFSTYTMAWFRQAPGKEREFVVAI KSSDNSTSYRDSVKGRFTISRDNAKSTMYLQMNSLKPEDTAVYYCAARREYSTIYTARYPGEYVYWGQGTQVTVSS <12D61/1-116, SEQ ID NO: 2261; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASRSIFSPNVVGWYRQAPGKQRELVAAV TSGGITNYADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCNARERGIYDSWGQGTQVTVSS <13G31/1-125, SEQ ID NO: 2262; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFSRYKMGWFRQAPGKEREFVAAS RWSGGIKYHADSVKGRFTISRDDAKNSIYLQMNTLKPEDTAVYYCAADDYLGGDNWYLGPYDSWGQGTQVTVSS <22C65/1-124, SEQ ID NO: 2263; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSGFLFDSYAMGWFRQAPGKEREFVAAI RWSGSATDYSDSVKGRFTISRDNAKNTVYLQMNSLIPEDTAVYYCAARKTYRSLTYYGEYDSWGQGTQVTVSS <11A71/1-125, SEQ ID NO: 2264; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASRSIRSVSVMGWYRLAPGNQRELVATI TADGITNYADSVKGRFTVSRDNGRNTVYLQMNSLKPEDTAVYYCNVDRLLYYSSGYYQTSVDVWGQGTQVTVSS <11B91/1-125, SEQ ID NO: 2265; PRT; -> EVQLVESGGALVQPGGSLRLSCAASGSIRSINTMGWYRQAPGNQREFVAAV TEGGTTSYAASVKGRFTISRDKAKNTVLLQMDSLKPEDTAVYYCNADRFLYYSAGRYDTGSDIWGQGTQVTVSS <11A81/1-125, SEQ ID NO: 2266; PRT; -> EVQLVESGGALVQPGGSLRLSCAASDSIRSINIMGWYRQAPGKQREFVAAV TEDGSINYAESVKGRFTISRDKAKNALYLQMNSLKPEDMAVYYCNADRVLYYSDSRYYTGSNYWGQGTQVTVSS <11B121/1-127, SEQ ID NO: 2267; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGSSASINTMGWYRQAPGEQRELVAEI TEGGIINYTDSVKGRFTISRDNAKNTVYLEMNNLKPEDTAVYYCNADRALYRNYSDGRYYTGYDYWGQGTQVTVSS <12D31/1-115, SEQ ID NO: 2268; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASRNIFDFNDMGWYRQGPGKEREFVALI NVGGVAKYEDSVKGRFTISRDNAENTVYLQMNNLKPEDMAVYYCNARILSRNYWGQGNQVTVSS <11B51/1-127, SEQ ID NO: 2269; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFSGRGMGWFRQAPGKEREFVAAV SWSGGNTYYADSVKGRFTISRDNAKSTVYLQMDSLKPEDTAVYYCAASRRFYSGLYYYTDDAYEYWGQGTQVTVSS <13G51/1-127, SEQ ID NO: 2270; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASGGTFNGRAVGWFRQAPGEEREFVTGI SWSGGSTDYADSVKGRFTISRDNSKNTVSLQMNSLKPEDTAVYYCAASRRFYSGLVYYSVDAYENWGQGTQVTVSS <13F82A/1-130, SEQ ID NO: 2271; PRT; -> EVQLVESGGGLVQAGGSLRLSCAISGRTLSGRAMGWFRQAPGKEREFREF VAATSWSGGSKYVADSVTGRFTIFRDNAENTAYLQMNSLNPEDTAVYYCAVTKRYYSIKYYSTVEDYEYWGQGTQVTVSS <13E101/1-128, SEQ ID NO: 2272; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSGRTFNNDHMGWFRQAPGTERELVAAT GRRGGPTYYADSVKGRFTISRDNAESTVYLQMNSLKAEDTAVYYCAANRYYCSTYGCLSTPRQYDYWGQGTQVTVSS <22B85/1-120, SEQ ID NO: 2273; PRT; -> EVQLVESGGGLVRPGGSLRLSCATSGSDIGINAMGWYRQAPGNQRELVATI TGSTGTTYADSVKGRFAISRDGAKNTVYLQMDSLKPEDTAVYYCNLRVYTGTYGGRNYWGQGTQTVSS <11B12/1-118, SEQ ID NO: 2274; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRALINYAMGWFRQAPGKEREFVSAI NWSGSHTDYGDSVKGRFAISRDNAKNTVYLQMHSLKPEDTAVYHCATGYSLPAFDSWGPGTQVTVSS <13G61/1-118, SEQ ID NO: 2275; PRT; -> EVQLVESGGGVVQAGGSLRLSCAPSGRTFSSYVMGWVRQAPGKAREFVAGI TRNSGRTRYADSVKGRFTISRDNADNTVTLQMNSLKPEDTAVYYCAGGIDLYTFHYFGQGTQVTVSS <14H41/1-118, SEQ ID NO: 2276; PRT; -> EVQLVESGGGLVQAGGSLRLSCAPSGRTFSSYVMGWVRQAPGKAREFVAGI TRNSIRTRYADSVKGRFTISRDNADNTVTLQMNSLKPEDTAVYYCAGGIDLYTFDYFGQTQVTVSS <11B81/1-126, SEQ ID NO: 2277; PRT; -> EVQLVESGGGLVQGGSLRLSCAASGRPVNNYIMGWFRQALGQGREFVAAI NRNGATAAYADSVKGRFTISRDNAEDLLYLQMNLLKPEDTAVYYCAANSDSGFDSYSVWAAYEYWGQGTQVTVSS <11C11/1-121, SEQ ID NO: 2278; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSAYAMGWFRQAPGKERESVATI RWTGGSSSTSYADSVKGRFTISKNTAENTVYLQMNSLKPEDTAVYYCAVLLTVWDTYKYWGQGTQVTVSS <12D92/1-123, SEQ ID NO: 2279; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTYNMAWFRQAPGKEREFVAAMNWS GGSTKYAESVKGRFTISRANDNNPLYLQMNTLKPEDTAVYYCAATNRWYTGVYDLPSRYEYWGQGTQVTVSS <13E61/1-123, SEQ ID NO: 2280; PRT; -> EVQLVESGGGLVQAGGSLRLSCTASGQTFNMGWFRQAPGKEREFVAAISWS QYNTKYADSVKGRFTISRDNAINSLYLQMDTLKPEDTAVYYCAATNRWFSAVYDLPSRYTYWGQGTQVTVSS <22B71/1-114, SEQ ID NO: 2281; PRT; -> EVQLVESGGAFVQGGSLRLSCAASGSDVWFNVMGWYRQGPGQQLELVASI TYGGNINYGDPVKGRFSISRDNALKTVYLQMNSLKPEDTAVYYCYADLPSRLWGQGTQVTVSS <21A121/1-123, SEQ ID NO: 2282; PRT; -> EVQLVESGGGLVQAGGSLRLSCTASGRAFNMGWFRQAPGKEREFVAGVNWG GGSTKVADSVKERFTISRDYDNSPVYLQMNTLKPEDTAVYYCAATSRWYSAVYDLPTRYDYWGQGTQVTVSS <13F101/1-124, SEQ ID NO: 2283; PRT; -> EVQLVESGGGLVQAGGSLRLSCQLSGGTVSDLHMGWFRQAPGKEREFVGFT RWPSITYIAEHVKGRFTISRDNAKNTVYLQMNSLEREDTAVYYCAADRSYSIDYRHADSYSYWGQGTQVTVSS <11A43/1-123, SEQ ID NO: 2284; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSIFRVNHMGWYRQAPGKQREFVAAI TSDHITWYADAVKGRFTISRDNAHNTVTLQMNSLRPEDTAVYYCAADPLLFYGVGSADVDYWGQGTQVTVSS <12C81/1-117, SEQ ID NO: 2285; PRT; -> EVQLVESGGGLVQPGGSLRLSCAGSGNIVRDNTMAWYRQAPGNQRDLVATI NVGGGTYYAGPVKGRFTISRDNAKNSVYLQMNSLKPEDTSVYYCNVISGLVQRDYWGQGTQVTVSS <11B21/1-124, SEQ ID NO: 2286; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSMYLMGWFRQAPGKEREFVSTI NRRGGNTYYADSVKGRFTISRDNARNTVYLQMNSLKPEDTAVYYCAAGGHLLGYDVQWEPDYWGQGTQVTVSS <11B71/1-126, SEQ ID NO: 2287; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFERYAMGWFRQAPGKEREFVATI SWSGGRDTVYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAHKRTYELGAHSTDFGSWGQGTQVTVSS <12C121/1-126, SEQ ID NO: 2288; PRT; -> EVQLVESGGDLVQPGESLRLSCAVSGVTVDYSGIGWFRQAPEKEREAVSCI ESGDCTTTYVDSVKGRFTISRDNAKNAVYLQMNSLKPEDTGVYYCATAVFVDSGDFSVCRGVGYWGKGTQVTVSS <22C51/1-121, SEQ ID NO: 2289; PRT; -> EVQLVESGGGLVQAGASLRLSCAASGRTFSRYDIGWFRQAPGKGREFVAAI NWSGGTTSFGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAALRSWPRGVDSGSWGQGTQVTVSS <12D11/1-123, SEQ ID NO: 2290; PRT; -> EVQLVESGGGLVQTGGSLRLSCAASGRTFSGSRMGWFRQAPGKEREFVAAI RWSGGITWYAESVKSRFTISRDNTKNTIDLQINSLKPEDTAVYYCAADVIYKNIGSGSFDYWGQGTQVTVSS <12D14/1-123, SEQ ID NO: 2291; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSGSRMGWLRQAPGKEREFVAAV RWSGGITWYAESVKGRFTISRDNTKNTIDLQINSLKPEDTAVYYCAADVIYKNIGSGSFDYWGQGTQTVSS <12C111/1-123, SEQ ID NO: 2292; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSGLTFSSYAMGWFRQAPGKVREFVATI SRSGGRTSYADSVKGRFIVSRDNAKNTADLQMNDLKPEDTAVYYCGASKWYGGFGDTDIEYWGQGTQVTVSS <22B55/1-123, SEQ ID NO: 2293; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSGLTFSTYAMGWFRQAPGKVREFVATI SRSGGRTSYADSVKGRFIVSRDNAKNTADLQMNELKPEDTAVYYCGASKWYGGFGDTDIEYWGQGTQVTVSS <14H121/1-113, SEQ ID NO: 2294; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAMGWFRQGPGKRRELVARI AGGSTNYADSVKGRFTISRDDAKNTVFLQMNSLKPEDTAVYYCNVDGPFGNWGQGTQVTVSS <12C71/1-125, SEQ ID NO: 2295; PRT; -> EVQLVESGGGLNQAGGSLRLSCTASGGTFGSYALGWFRQSPGKERESVAAI DWDGSRTQYADSVKGRFTISRENVKDTMYLQMNSLQAEDTGVYYCVRSRHSGNTLSFSLKYDYWGQGTQVTVSS <21A31/1-125, SEQ ID NO: 2296; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASEPTFSSVAMGWFRQGPGKEREFAATI TWSGDSTYVTDSVKGRFTISRDNARNTAYLQMDSLRPEDTAVYSCAARRWSGTLSLFDNEYYYWGQGTQVTVSS <12C91/1-121, SEQ ID NO: 2297; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGRTSSYYHMAWFRQAPGKEREFIAAI NLSSGSTYYPDSVKGRFTISRGNAKNTVNLQMNSLKPEDTAVYYCAADNYRDSYLEYDYWGQGTQVTVSS <14H91/1-125, SEQ ID NO: 2298; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASGRTFSNYRMAWFRQAPRKEREFVAAI SRSGESTYFADSMKGRFTISRDNTESTGYLQMNNLKPEDTAVYYCAASWDHGDYVDGGFFYDYWGQGTQVTVSS <12C42/1-124, SEQ ID NO: 2299; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMHWFRQAPGSERDFVAGI SWDGGSTFYANSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAAAGSAGPPSIDRQYDYWGQGTQVTVSS <12D102/1-118, SEQ ID NO: 2300; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGSSLSFNAMGWSREAPGKRRELVARI ISDDSTLYADSVKGRFTISRDYAKNTAYLQMNSLKPEDTAVYYCVADVRDSIWRSYWGQGTQVTVSS <11A52/1-120, SEQ ID NO: 2301; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRALSNYAMRWFRQAPGKEREFVATI NWSGSHTDYADSVKGRFTISRDNAENTVYLQMNSLTPEDTAVYYCASGWGATQAQSGFWGQGTQVTVSS <14H111/1-120, SEQ ID NO: 2302; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRALISFAMRWFRQAPGKEREFVAAI NWSGTHTDYADSVKGRFTISRDNAENTVYLLMNSLIPEDTAVYYCATGWGATQAQHGFWGQGTQVTVSS <11B61/1-120, SEQ ID NO: 2303; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTSSGYGMGWFRQAPGKEREFVAAV GWYGSTYFADSVKGRFTIYRDNAQNTMYLQMNSLKPEDTAVYYCAASSSLATISQPSSWGQGTQVTVSS <12E42/1-118, SEQ ID NO: 2304; PRT; -> EVQLVESGGGLVQPGGSLRLSCAHSGRAFSLRTMGWYRQAPGNQRELVALI SAGDSTYYPDSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAKAVTSRDHEYWGQGTQVTVSS <13F81A/1-128, SEQ ID NO: 2305; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVAAI SWTGGSSYYGDSVKGRSTISRENAENTVYLQMNSLKPEDTAVYYCAANS DEFYSGTLKLQSRMVEYWGQGTQVTVSS <11B102/1-118, SEQ ID NO: 2306; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGIFSSHAISWFRQAPGKAREFVA AINWSGSHRDYADSAKGRFTISRDNAKKTAYLQMNSLRPEDTAVYYCVGGWKTDEYVKWGQGTQVTVSS <21A41/1-120, SEQ ID NO: 2307; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRIFSNYAWSWFRQAPGKERGFVA AINWSGGYTDYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAVYYCRPGWVTPSYEYGNWGQGTQVTVSS <14H101/1-128, SEQ ID NO: 2308; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFISSPMGWFRQAPGKEREVVA ATTRSGGLPYYSDSVKGRFTISRDNAKNTVDLQMSSLKPEDTAAYYCAADQKYGMSYSRLWLVSEYEYWGQGTQVTVSS <12E21/1-115, SEQ ID NO: 2309; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGSIDSIHVVGWYRKAPGKQREVVA YIGTAGATHYADSVKGRFTISRDNAENLVYLQMNNLKPEDTAVYYCSAGWGDSAYWGQGTQVTVSS <13F21/1-123, SEQ ID NO: 2310; PRT; -> EVQLVESGGGLVQSGGSLRLSCVASGTIVSINATSWYRQAPGNQRELVA TIIGDGRTHYADSVKDRFTISRDAAANLVYLQMNSLKPSDTAIYSCNANGIESYGWGNRHFNYWTVGTQVTVSS <12E33/1-119, SEQ ID NO: 2311; PRT; -> EVQLVESGGGMVQAGGSLRLSCAASGLTLSNYGMGWFRQAPGKEREFVS SINWSGTHTYDADFVKGRFIISRDNAKNTVYLQINSLKPEDTAVYYCAAGGWGTGRYNYWGQGTQVTVSS <13G11/1-122, SEQ ID NO: 2312; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFISNYAMGWFRQAPGKEREFV ATINWSGSHSDYADSVKGRFTISRDNAKNTVYLQMNNLKSEDTAVYYCAPGWGTAPLSTSVYWGQGTQVTVSS

TABLE B-3 Nanobodies against HER2 obtained as described in Example 4 <Name, SEQ ID #; PRT (protein); -> <118N121_A1_4_OK/1-127, SEQ ID NO: 2113; PRT; -> EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAAAW FRQSPGKERDLVAGIMWDGRSLFYADSVKGRFTISRDNAKNTLHLQMNS LKPEDTAVYYCAYHKTPYTTLELNRPHAFGSWGQGTQVTVSS <118N121_A6_2_OK/1-123, SEQ ID NO: 2313; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGRTFSGYSVGW FRQSPGKEREFVGGINWSGRTYYVDSVKGRFTFSRDNAKNTVYLQMNSLKPEDT AIYLCAVDRFNTIANLPGEYDYWGQGTQVTVSS <118N121_B8_1_OK/1-135, SEQ ID NO: 2314; PRT; -> EVQLVESGGGLVQDGGSLRLSCAASGQLANFASYAMGWFRQAPGKA REFVAAIRGSGGSTYIADPARSTYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCACETFN SISNLPGEYDYWGQGTQVTVSS <118N121_A2_2_OK/1-124, SEQ ID NO: 2315; PRT; -> KVQLVESGGGLVQAGGSLRLSCAASGRTFSNYSVGWFRQAPGKEREFVA ALSKDGARTYYAASVKGRFTIYRDNAKNVVYLQMSVLNGEDTAVYYCAADHFTFMSNLPSEYDYWGQGTQVTVSS <118N121_A8_2_OK/1-124, SEQ ID NO: 2316; PRT; -> EVQLVESGGGLVQAGGSLTLSCVISGLTLESHAMGWFRQAPGEEREF VATIRWSGSATFYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARKIYRSLSYYGDYDSWGQGTQVTVSS <118N121_B3_1_OK/1-123, SEQ ID NO: 2317; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGRTFSDLALGWFRRAPGKERE HVAAISSSGVTTIYADSVRGRFTISRDEAKNTVYLEMNSLKTDDTAVYYCAARLTMATPNQSQYYYWGQGTQVTVSS <118N121_A5_2_OK/1-114, SEQ ID NO: 2318; PRT; -> EVQLVESGGGSVQPGGSLRLSCVASGSISSTNAMGWHRQVSGKERE LVAIVTDGFTNYADFAKGRFTISRDNAKTTVYLQMNSLQPEDTARYYCRYSGIGTDNWGQGIEVTVSS <118N121_A9_2_OK/1-114, SEQ ID NO: 2319; PRT; -> EVQLVESGGGSVQPGGSLRLSCVASGSISSVNAMGWHRQVPGKQRE LVAIVTDGFTNYADFAKGRFTISRDNAKTTVYLQMNSLQPEDTARYYCRYSGIGTDNWGQGIEVTVSS <118N121_A7_1_OK/1-122, SEQ ID NO: 2320; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGNIKSIDVMGWHRQAPGKERE LVSDISFGGNTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYADILYKTDITYYRNDFWGQGTQVTVSS <118N121_A10_1_OK/1-131, SEQ ID NO: 2321; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGFSFADYAIGWFRQAPGKEREGVSCIANSE GTKYYADSAQGRLPISSDNAKKTVYLQMDSLKPEDTAVYYCAALPYTICPVVVKKGAVYYGVDDYWGKGTQVTVSS <118N121_A11_1_OK/1-120, SEQ ID NO: 2322; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFPFGMYGMRWVRQAPGKGPERVSSINSDG DTTYYADSVKGRFTISRDNDENMLYLQMNSLKPEDTAVYYCATGFSDRSFAVTHKGQGTQVTVSS <118N121_B7_4_OK/1-124, SEQ ID NO: 2323; PRT; -> EVQLVESGGGLEQAGGSLRLSCAASGLTFRSAAMGWFRQGPGKEREFVAAISRDG AATYYTDSVKGRFTISRDNAKNTVFLQMNSLKPEDTAIYYCAADFRLARLRVADDYDYWGQGTQVTVSS <118N121_B2_1_OK/1-130, SEQ ID NO: 2324; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGFSLDDRAIAWFRQAPGKAREGVSCITPHH GGIIFTRESVKGRFATSSDSAKNTVYLQMHSLKPEDTAVYYCATLRTDYSINWANCQRDSLYGYWGQGTQVTVSS <118N121_B7_1_OK/1-119, SEQ ID NO: 2325; PRT; -> EMQLVESGGGLVQPGGSLRLSCAASGNIPPINAMAWYRQAPGNERELVAAVTSGG GTNYATSVKGRFIISRDDSKNTVDLQMNSLKPEDTAVYYCNLGGWTRTHPFDYWGQGTQVTVSS

TABLE B-4 Bivalent Nanobodies against HER2 as described in Example 12 <Name, SEQ ID #; PRT (protein); -> <2A4-9GS-2A4, SEQ ID NO: 2326; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSA INWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGW QSTTKNQGYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLS CAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTIS RDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS <2A5-9GS-2A5, SEQ ID NO: 2327; PRT; -> EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSS INWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNW GDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGS LRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGR FTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQG TQVTVSS <2C3-9GS-2C3, SEQ ID NO: 2328; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSS INWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGW KIVPTDRTGHGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCV ASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRD NAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS <2D3-9GS-2D3, SEQ ID NO: 2329; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSS INWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNW RDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGSLVQPGGS LRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGR FTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQG TQVTVSS <5F7-9GS-5F7, SEQ ID NO: 2330; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVAL ISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRT AAQGTDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCA ASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDN AKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS

TABLE B-5 Bispecific Nanobodies against HER2 and against serum albumin as described in Example 12 <Name, SEQ ID #; PRT (protein); -> <2C3-9GS-ALB1, SEQ ID NO: 2331; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSS INWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGW KIVPTDRTGHGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCA ASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <2A4-9GS-ALB1, SEQ ID NO: 2332; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSA INWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGW QSTTKNQGYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLS CAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <2A5-9GS-ALB1, SEQ ID NO: 2333; PRT; -> EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSS INWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNW GDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNS LRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <2D3-9GS-ALB1, SEQ ID NO: 2334; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSS INWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNW RDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNS LRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <5F7-9GS-ALB1, SEQ ID NO: 2335; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVAL ISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRT AAQGTDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCA ASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS

TABLE B-6 Biparatopic Nanobodies against HER2 <Name, SEQ ID #; PRT (protein); -> <27B3-35GS-2D3, SEQ ID NO: 2336; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDN AKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTIS RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27C3-35GS-2D3, SEQ ID NO: 2337; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRD NAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYAD SVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27E5-35GS-2D3, SEQ ID NO: 2338; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDN AKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTIS RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27F2-35GS-2D3, SEQ ID NO: 2339; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTLAWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTISISRD NVEYTVDLQMDSLKPEDTAVYYCRDINGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISR NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D6-35GS-2D3, SEQ ID NO: 2340; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNA KNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <30D10-35GS-2D3, SEQ ID NO: 2341; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNA KNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <47D5-35GS-2D3, SEQ ID NO: 2342; PRT; -> KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNA KDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRF TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <DUMMY-35GS-2D3, SEQ ID NO: 2343; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDN AKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYA DSVNGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <2D3-35GS-2D3, SEQ ID NO: 2344; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <DUMMY-35GS-47D5, SEQ ID NO: 2345; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDN AKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSAD SVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS <5F7-35GS-47D5, SEQ ID NO: 2346; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNA KNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTI SRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS <47D5-35GS-5F7, SEQ ID NO: 2347; PRT; -> KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNA KDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS <2D3-35GS-47D5, SEQ ID NO: 2348; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSV KGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS <27F7-35GS-2D3, SEQ ID NO: 2349; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSTIRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDN AKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRN NANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <28F6-35GS-2D3, SEQ ID NO: 2350; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAK NTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNA NNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <28G3-35GS-2D3, SEQ ID NO: 2351; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAK NTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNA NNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <28G5-35GS-2D3, SEQ ID NO: 2352; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNA NNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGSGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <29D9-35GS-2D3, SEQ ID NO: 2353; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTMAWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTVSRDNA KNTVYLQMNSLKAEDTAVYYCWDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <29E9-35GS-2D3, SEQ ID NO: 2354; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNA KNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <30E10-35GS-2D3, SEQ ID NO: 2355; PRT; -> KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <31D11-35GS-2D3, SEQ ID NO: 2356; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAK NTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGSGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNA NNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27G2-35GS-2D3, SEQ ID NO: 2357; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNN ANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <P27G4-35GS-2D3, SEQ ID NO: 2358; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNA NNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27G5-35GS-2D3, SEQ ID NO: 2359; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNA KNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27G7-35GS-2D3, SEQ ID NO: 2360; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNA NNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H1-35GS-2D3, SEQ ID NO: 2361; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNA NNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H2-35GS-2D3, SEQ ID NO: 2362; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNA NNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H3-35GS-2D3, SEQ ID NO: 2363; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H4-35GS-2D3, SEQ ID NO: 2364; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFTISRDNA KNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTI SRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H5-35GS-2D3, SEQ ID NO: 2365; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGIGWFRQASGKEREGVSCITSSDGSTYYADSVKGRFTISSDN AKNTVYLQMNSLKPEDTAVYYCAALPFVCPSGSYSDYGDEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLNESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTD YADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDACTTWFEKSGSAGQGTQVTVSS <27H7-35GS-2D3, SEQ ID NO: 2366; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNA ENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27A3-35GS-2D3, SEQ ID NO: 2367; PRT; -> EVQLVESGGGLVQAGGSLSLSCVASGRFFSTRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNA KNTVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27A4-35GS-2D3, SEQ ID NO: 2368; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNA NNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27A5-35GS-2D3, SEQ ID NO: 2369; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAK NTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNA NNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27B1-35GS-2D3, SEQ ID NO: 2370; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27B2-35GS-2D3, SEQ ID NO: 2371; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPSIRAIAWYRQAPGKQRDWVATSGTGYGATYDDSVKGRFTLSRDNAK NTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNA NNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27B5-35GS-2D3, SEQ ID NO: 2372; PRT; -> EVQLVESGGGLVQAGGSLRLPCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNA ENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQTVSS <27B7-35GS-2D3, SEQ ID NO: 2373; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISSGGGSITTYADSVKGRFTISRD NAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27C2-35GS-2D3, SEQ ID NO: 2374; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27C5-35GS-2D3, SEQ ID NO: 2375; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAK NTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNA NNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27C7-35GS-2D3, SEQ ID NO: 2376; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNA DNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D1-35GS-2D3, SEQ ID NO: 2377; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDN AKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGIHTDYADSVKGRFT ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D2-35GS-2D3, SEQ ID NO: 2378; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRD NAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISR NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D3-35GS-2D3, SEQ ID NO: 2379; PRT; -> EVQLMESGGGLVQFGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRD NAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISR NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D4-35GS-2D3, SEQ ID NO: 2380; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D7-35GS-2D3, SEQ ID NO: 2381; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDN AKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFT ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27E2-35GS-2D3, SEQ ID NO: 2382; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDN AKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFT ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27E4-35GS-2D3, SEQ ID NO: 2383; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFTISRDNA KNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTI SRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27E7-35GS-2D3, SEQ ID NO: 2384; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKERELVATILSEGDTNYVDPVKGRFTISRDNA KNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTI SRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <29H1-35GS-2D3, SEQ ID NO: 2385; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <30H9-35GS-2D3, SEQ ID NO: 2386; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <39C1-35GS-2D3, SEQ ID NO: 2387; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27G8-35GS-2D3, SEQ ID NO: 2388; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDN AKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFT ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <29H2-35GS-2D3, SEQ ID NO: 2389; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDN AKNTLFLQMNNLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFT ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQTVSS <38C6-35GS-2D3, SEQ ID NO: 2390; PRT; -> EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDN AKNTLFLQMNSLSPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFT ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS

TABLE C-1 Sequence of HER2 binding Nanobodies aligned by family HERCEPTIN ® COMPETING 13D11 EVQLVESGGGLVHPGGSLRLSCVGSGFSLDDYGMTWVRRAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCGQGWKIVPTNPRGHGTQVTVSS  2B4 EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS  2G2 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYTDPVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNRGWKIVPTDLGGHGTQVTVSS 13D2 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLRSEDTAVYSCNQGWKIVPTDRGGHGTQVTVSS  2D5 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS  2F4 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRRGHGTQVTVSS  2C3 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS 17E3 EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVA SIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLTPEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS 17H3 EVQLMESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS 17D2 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS  2F1 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKELEWISSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIVPMDRRGHGTQVTVSS  2E2 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS  2C2 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNARNTLFLQMNSLTPEDTAIYYCNQGWKILPTDRRGHGTQVTVSS  2E3 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS 13B10 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGFEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS  2D1 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNRGWKILPTNRGSHGTQVTVSS  2H3 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS  2H1 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVRGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS  2C1 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 15C5 EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWNVTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS  2B3 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDCADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 29H2 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 17E4 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 17A2 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNKGWKVMPTDRGTHGTQVTVSS 15D1 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKVWPTDRGTHGTQVTVSS 17B8 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS 15C11 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTPVTVSS 15G8 EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPENTAVYYCNQGWKILPAERRGHGTQVTVSS 17H4 EVQLVESGGGLVQPGGSLRLSCVASGFSLIMYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLHMNNLSPEDTAVYYCGQGWKIHPADRGGHGTQVTVSS 27G8 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS 38C6 EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS  2A4 EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS 15G7 EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTAVYYCATGWQSTTKNQGYWGQGTQVTVSS 15B7 EVQLVESGGGLVQPGGSLKLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS  5G4 EVQLVESGGGLVQPGGSLTLSCAGSGFIFDDYAMSWVRQAPGKGLEWVSSINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTAVYYCATGWQSTTKNQNYWGQGTQVTVSS 13B2 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHKDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS  2E5 EVQLVESGGSLVQPGESLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 15G1 EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 27B1 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 17E7 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 17D8 EVQLVESGGSLVPPGGSLRLSCAVSGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNMLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS  5F8 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYALSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS  2D4 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS 13D8 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 17G8 EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS  2H4 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS  2F3 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS  2F5 EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 30E10 KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 29H1 EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 17E2 EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS  2B1 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS  2A5 EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 13C12 EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 17E10 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 27D4 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 15F9 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 30H9 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 39C1 EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 27G2 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS  2D3 EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS  5F7 EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS PBMP118N121_A1_4_OK/1-127 EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAAAWFRQSPGKERDLNAGIMWDGRSLFYADSVKGRFTISRDNAKNTLHLQMNSLKPEDTAVYYCAYHKTPYTTLELNRPHAFGSWGQGTQVTVSS OT-FPB COMPETING 47D5 KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS HER2 BINDING 14B11 EVQLNESGGGLVQAGGSLRLSCAASGSTFSSYGMGWFRQVPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTARYYCGVETYGSGSSLMTEYDYWGQGTQVTVSS 14B10 EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS 14B4 EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPEDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS 14C11 EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGATAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS 14B5 EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETFGSGSSLMSEYDYWGQGTQVTVSS 14C6 EVQLVESGGGSVQAGGSLRLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATDTYGSGSSLMNEYDYWGQGTQVTVSS 14A4 EVQLVESGGGSVQAGSSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS 14B3 EVQLVESGGGLVQPGGSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS 14C1 EVQLVESGGGSVQAGGSLRLSCAASGSTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATETYGSGSSLMNEYDYWGQGTQVTVSS 14A12 EVQLNKSGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS 14A2 EVQLVESGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS 14A1 EVQLVESGGGSVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDYWGHGTQVTVSS 17C3 EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS 46D3 KVQLVESGGGLVQAGGSLRLSCAASGRTFTEYSMGWFRQAPGKEREFVATISWNYGYTYYSDSVKGRFTVSRDIAENTVYLQMNTLKSEDTAVYYCAAKIGWLSIRGDEYEYWGQGTQVTVSS 27H5 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGIGWFRQASGKEREGVSCITSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAALPFVCPSGSYSDYGDEYDYWGQGTQVTVSS 17C2 EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMSWVRQAPGKGLEWVSAVDSGGGRTDYAHSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCTKHVSDSDYTEYDYWGQGTQVTVSS 17D11 EVQLVESGGGLVQAGGSLRLSCTASGRTSSTSAMGWFRQAPGKEREFVATISRGGSATYYADLKKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAARRSSLYTSSNVFEDYWGQGTQVTVSS 15A6 EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS 17B6 EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTMAWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADYWGQGTQVTVSS 17C5 EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTPVTVSS 15E11 EVQLVESGGGLVQAGGSLRLSCVASRIPFSSRTMAWYRQAPGKQRDWVATISARGMPAYEDSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCRDVNADYWGQGTQVTVSS 15C2 EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAQGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS 2A3 EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQAPGKPRDWVA TIRNGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTATYYLCRDVNGDIWQGTQVTVSS 27A5 EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVA TIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSS  2C5 EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQTPGKSRDWVA TIRSGTPVYADSVKGRFTVSRDNAKNTLYLRMNSLKSEDSATYTCRAVNADIWGQGTQVTVSS 27G5 EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS 13A9 EVQLVESGGGLVQAGGSLRLSCVASRIPASIRTMAWYRQAPGKQRDWVATIGTGGTPAYADSFKGRFTVSRDNANHTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS 29E9 EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS 15D8 EVQLVESGGGLVQPGGSLKLSCVASTIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS 15G4 EVQLVESGGGLVQAGGSLRLSCVASGIPFRSRTMAWYRQAPGKTRDWVATIGTHGTPLYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCWDVNGDYWGQGTQVTVSS 15D12 EVQLVESGGGLVQAGESLRLSCATSGITFKRYVMGWYRQGPGKQRELVATVNDGGTTSYADSVKGRFAISRDNAKNTAYLQMNSLKAEDTAVYYCNAVWKLPRFVDNDYWGQGTQVTVSS 15E12 EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS 13D7 EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS 13A8 EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV YADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 15A4 EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV YADSMKGRFTISRDNAKNTVYLQINSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 17F7 EVQLVESGGGLVQAGGSLRLSCVASGIAQS  IRVMAWYRQPPGKQRDWVGTISSDGTAN YADSVKGRFTISRDNAKKTMYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS 15C8 EVQLVESGGGLVQAGGSLRLSCAASGIAFR  IRTMAWYRQAPGKQRDWVATSDSGGTTL YADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 17A10 EVQLVESGGGLVQAGGSLRLSCVASGIPSI   RAIAWYRQAPGKQRDWVATSGTGYGAT YDDSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 27D3 EVQLMESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV YADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 13B12 EVQLVESGGGLVQAGGSLRLSCAASGIAFR  IRTMAWYRQAPGKQRDWVATIGSDGTTI YADSVKGRFTLSRHNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 15B2 EVQLVESGGGLVQAGGSLRLSCVVSGIPSS  IRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS 15B11 EVQLVESGGGSVQAGGSLRLSCVVSGIPSS  IRAMAWYRQAPGRQRDWVATIYSRSGGAVYADSVKGRFTISSDNAKNTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS 13C9 EVQLVESGGGLVQAGGSLRLSCVASGIPSI  HAMAWYRQAPGKQRDWGATTYSRGG  TTYNDSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 17D5 EVQLVESGGGLVQPGGSLRLSCAASGIIGT IRTMAWYRQAPGKQRDWVA  SIGTRGAPVYADSVNGRFTISRDGATNTVFLQMNNLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 27B5 EVQLVESGGGLVQAGGSLRLPCAASGIAFR IRTMAWYRQAPGKQRDWVA  TSDSGGTTLYADSVKGRETVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 27C7 EVQLVESGGGLVQAGGSLRLSCAASGIAFR IRTMAWYRQAPGKQRDWVA  TSDSGGTTLYADSVKGRFTVSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 13D4 EVQLVESGGGLVQAGGSLRLSCVVSGIPSS IRAMAWYRQAPGRQRDWVA TIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLEPDDTAVYYCRDVNREYWGQGTQVTVSS 15G5 EVQLVESGGGLVQAGGSLRLSCVVSGIPST IRAMAWYRQAPGRQRDWVA TIYSPSGSAVYADSVKGRFTISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS 13C4 EVQLVESGGGLVQAGGSLRLSCVVSGIPSS IRAMAWYRQAPGRQRDWVA TIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS 46G1 EVQLVESGGGLVQAGGSLRLSCAASGRTFSDDAMGWFRQAPGKERECVASLYLNGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSQYNYWGQGTQVTVSS 46E4 EVQLVESGGGLVQAGGSLRLSCAASGRAFKDDAVGWFRQAPGKERECVASMYLDGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSEYNYWGQGTQVTVSS 17B5 EVQLVESGGGLVQTGGSLRLSCAASGSTFRTDMMGWYRQAPGKQREFVASITKFGSTNYADSVKGRFTISNDNAKDTVYLQMNSLKSEDTAVYYCRNFNRDLWGQGTQVTVSS 15C9 EVQLVESGGGLVQAGGSLKLSCVNSGIPSTLRAMAWYRQAPGRQRDWVATSSNTGGTTYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCRDVNRDLWGQGTQVTVSS 13D10 EVQLVESGGGLVQPGGSLRLSCAASSVITLDSNAIGWFRQAPGKEREEVSCIASSDGSTYYAESVKGRFTISKDYTRNTVYLQVNSLKPEDTAVYHCATDANPNCGLNVWNSWGQGTQVTVSS 17C6 EVQLVESGGGLVQAGGSLTLSCAASGSTSSLDIMAWYRQAPEKQRELVASVSGGGNSDYASSVKGRFTISGDTAKSTLYLQMNSLKPEDTAMYYCYGRDYYYMPFWGQGTQVTVSS 15A2 EVQLVESGGGLAQAGGSLSLSCAASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS 17A8 EVQLVESGGGLVQAGGSLSLSCAASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNMVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS 15G10 EVQLVESGGGLVQAGGSLSLSCAASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS 27A3 EVQLVESGGGLVQAGGSLSLSCVASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS 17H10 EVQLVESGGGLVQAGGSLSLSCSASGRFFS  TRVMAWYRQTPGNQREFVATIHSSGSTIYADSVRGRFAISRDNAKNTVYLQMRSLKPEDTAVYYCRDINADQWGQGTQVTVSS 30D10 EVQLVESGGGLVQAGGSLTLSCTASETTVR  IRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS 15H4 EVQLVESGGGLVQAGGSLTLSCAPSESTVS  FNTVAWYRQAPGEQREWVATISRQGMSTYPDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCRDINHDIWGRGSQVTVSS 17B7 EVQLVESGGGLVQAGGSLRLSCAASGIISS  FRTMAWYRQAPGKQRDWVATIGSDGLANYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYFCRDINRDYWGQGTQVTVSS 15D2 EVQLVESGGGLVQAGGSLRLSCVVSGVFGP  IRAMAWYRQAPGKQRDWVATIGSSGHPVYTDSVKGRFTFSKDGAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS 17G5 EVQLVESGGGLVQPGGSLRLSCAASGIGIAFSSRTMAWYRQAPGKQRDWVATIGSGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS 15B6 EVQLVESGGGLVQPGGSLRLSCAASGIIGS  FRTMAWYRQAPGNQRDWVATIGSRGLASYADSVRGRFTLSRDNAKKTVYLQMNSLKPEDTAIYYCRDINGDYWGQGTQVTVSS 27F2 EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTLAWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTISISRDNVEYTVDLQMDSLKPEDTAVYYCRDINGDYWGQGTQVTVSS 17F5 EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSS 17B2 EVQLVESGGGLVQPGGSLRLSCAGSGFTFSNYAMTWVRQAPGKGLEWVSGVGGDGVGSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCTKDISTFGWGPFDYWGQGTQVTVSS 27H4 EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS 13A4 EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS  2A1 EVQLVESGGGLVQAGGSLRLSCVASKITFRRYIMDWYRQAPGKQRELVASINSDGSTGYTDSVKGRFTISRDNTKNTLDLQMNSLKPEDTAVYYCHGRWLEIGAEYWGQGTQVTVSS 15E10 EVQLVESGGGLVQAGGSLKLSCVASGITFFRYTMGWYRQAPGKERELVAEISSADEPSFADAVKGRFTISRDNAKNTVVLQMNGLKPEDTAVYYCKGSWSYPGLTYWGKGTLVTVSS 27E7 EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKERELVATILSEGDTNYVDPVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSS 47E5 EVQLVESGGGLVQAGGSLRLSCAASASIFGFDSMGWYRQAPGNERILVAIISNGGTTSYRDSVKGRFTIARDNAKNTVSLQMNSLKPEDTAVYYCNLDRRSYNGRQYWGQGTQVTVSS  2G4 EVQLVESGGGLVQAGGSLRLSCAASGNIFSHNAMGWYRQAPGKQRELVTYITINGIANYVDSVKGRFTISRDNTKNTMYLQMVSLKPEDTAVYYCNVGGREYSGVYYYREYWGQGTQVTVSS 14D4 EVQLVESGGGLVQAGDSLRLSCAASGRALDTYVMGWFRQAPGDGREFVAHIFRSGITSYASSVKGRFTISRDNAKNTVYLQMASLKPEDTAAYYCAARPSDTTWSESSASWGQGTQVTVSS 17A5 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYSMSWVRQATGKGLEWVSGISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLKSEDTAVYYCAKDLGNSGRGPYTNWGQGTQVTVSS 15D10 EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYRMYWVRQAPGKGLEWVSAIKPDGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCGVPGFGWTFSSWGQGTQVTVSS 13C2 EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFTISRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS 17G11 EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRWGWYRQAPGKQRELVAAIDDGGNTEYSDFVNGRFTISRDNPETAVHLQMNSLKLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS 17A3 EVQLVESGGGLVQAGGSLSLSCAASATLHRFDNNWYRQAPGKQRELVATIAHDGSTNYANSVKGRFTISRDNARDTLFLQMHALQPEDTAVYMCNLHRWGLNYWGQGTQVTVSS 27B7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS 17A6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISTDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS 17D7 EVQLVESGGGLVQPGGSLRLSCAASGFTLDYCAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGSGTCYADFGSWGQGTQVTVSS 46D4 EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYAEDMKGRFTISRDNAKKTLYLQMNSLQSEDTAVYYCAKGWGPAVTSIPVATLGTQVTVSS 27B3 EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS 27E5 EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS 27D6 EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS 30D10 EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS 47G11 EVQLVESGGGLVQPGGSLRLSCAASGRIFYPMGWFRQAPGKEREFVAAIGSGDIITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSRDYSRSRDPTSYDRWGQGTQVTVSS 27C3 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSS

TABLE C-2 k_(off) rate of different Nanobodies as measured in Biacore ID k_(off) (s⁻¹) 2A4 2.05E−03 2A5 1.42E−03 2A6 1.65E−03 2B1 1.55E−03 2C4 1.26E−03 2D2 1.61E−03 2D4 1.65E−03 2F2 1.65E−03 2F3 1.53E−03 2F5 1.57E−03 2G5 1.56E−03 2H4 1.61E−03 2B2 1.19E−03 2B3 1.25E−03 2B4 2.77E−03 2B5 1.15E−03 2C1 1.18E−03 2C2 4.12E−03 2C3 1.11E−03 2D1 1.27E−03 2D5 1.20E−03 2F1 1.77E−03 2F4 1.07E−03 2G1 1.23E−03 2G2 1.30E−03 2G3 1.20E−03 2H1 1.09E−03 2H2 1.18E−03 2H3 1.15E−03 2H5 1.21E−03

TABLE C-3 Overview of k_(d)/k_(off)-, k_(a)-, and K_(d)-values for binding of Nanobodies 2D3, 5F7 and 47D5 to HER2. Fusion of a dummy Nanobody at the N-terminal end of the Nanobodies 2D3 and 47D5 does not significantly impact on the binding characteristics of 2D3 or 47D5 respectively. Nanobody ID k_(off) (s⁻¹) K_(on) (1/Ms) K_(D) (nM) 2D3 1.48E−03 1.36E+06 1.09 Dummy-2D3 1.13E−03 1.16E+06 1.77 5F7 3.02E−04 1.02E+06 0.29 47D5 8.62E−04 3.86E+05 2.23 Dummy-47D5 8.69E−04 2.71E+05 3.21

TABLE C-4 Off-rate analysis of HER2-ECD on 2D3, 47D5 and 2D3-35GS-47D5 coated sensor chips Analyte Protein on sensor chip k_(off) (1/s)  100 nM Her2 ECD 2D3-47D5 8.07E−5  100 nM Her2 ECD 2D3 2.10E−3  100 nM Her2 ECD 5F7 2.56E−3 1000 nM Her2 ECD 2D3-47D5 5.45E−5 1000 nM Her2 ECD 2D3 1.51E−3 1000 nM Her2 ECD 5F7 1.31E−3

TABLE C-5 Oligonucleotide primers used for generation of Omnitarg light chain V_(L )+ C_(L )by overlap extension For-sequences SEQ ID NO Rev-sequences SEQ ID NO >For_LCrescuepAX51 2396 >Rev_LC1pAX51 2408 tgattacgccaagct TAATAACAATCCAGCGGCTGCCGTAG GCAATAGGTATTTCATGTTGAAAATC T >For_LC1pAX51 2397 >Rev_LC2_OT 2409 tgattacgccaagcttgcatgca ATCGCCGACGGACGCGCTCAGGCTAC aattctatttcaaggagattttc TCGGAGATTGCGTCATCTGGATGTCG aacatga GC >For_LC2pAX51_OT 2398 >Rev_LC3_OT 2410 gctggattgttattactcgcggc CCGGCTTCTGTTGATACCAAGCAACC ccagccggccatggccGACATCC CCGATAGATACGTCCTGACTTGCT AGATGACG >For_LC3_OT 2399 >Rev_LC4_OT 2411 GCGTCCGTCGGCGATCGCGTTAC CCGCTGAAACGGGAAGGCACACCGGT CATCACATGCAAAGCAAGTCAGG GTAACGATATGATGCGGAGTAAAT ACGT >For_LC4_OT 2400 >Rev_LC5_OT 2412 ATCAACAGAAGCCGGGCAAGGCT ATAGTAGGTGGCGAAGTCCTCTGGCT CCGAAATTGCTCATTTACTCCGC GCAGGCTAGAGATAGTCAGGGTAA ATCA >For_LC5_OT 2401 >Rev_LC6_OT 2413 TTCCCGTTTCAGCGGAAGCGGCT TACCGTACGTTTAATTTCCACTTTCG CGGGTACTGATTTTACCCTGACT TACCCTGGCCAAAGGTATACGGGT ATCT >For_LC6_OT 2402 >Rev_LCrescue_VL_OT 2414 TTCGCCACCTACTATTGTCAGCA TCGGAAGGCGGAAAG ATACTATATTTACCCGTATACCT TTGG >For_LC7_OT 2403 >Rev_LC7 2415 ATTAAACGTACGGTAGCTGCCCC ATACGACGCTGGCCGTACCACTTTTC TAGCGTGTTTATCTTTCCGCCTT AGCTGCTCGTCGGAAGGCGGAAAG CCGA >For_LC8 2404 >Rev_LC8 2416 CGGCCAGCGTCGTATGTTTACTG CCGGACTGCAGTGCATTATCCACTTT AATAACTTCTATCCGCGCGAAGC CCATTGGACTTTAGCTTCGCGCGG TAAA >For_LC9 2405 >Rev_LC9 2417 TGCACTGCAGTCCGGCAATTCTC GGTCAGGGTAGAGCTCAGTGAGTAAG AAGAATCCGTGACGGAACAAGAT TGCTATCTTTGCTATCTTGTTCCG AGCA >For_LC10 2406 >Rev_LC10 2418 AGCTCTACCCTGACCTTGTCAAA GAAAGTCCCTGATGGGTCACTTCACA GGCAGATTATGAAAAACACAAAG GGCGTAAACTTTGTGTTTT TTTA >For_LC11 2407 >Rev_LC11 2419 CCATCAGGGACTTTCGAGTCCGG aaatagaattggcgcgccttattaGC TTACAAAGTCTTTTAACCGCGG ACTCACCGCGGTTAAAAGAC >Rev_LCrescue 2420 aaatagaattggcgc

TABLE C-6 Oligonucleotide primers used for generation of Omnitarg heavy chain V_(H )+ CH₁ by overlap extension SEQ ID SEQ ID For NO Rev NO >For_HCrescue 2421 >Rev_HC1 2434 gtgctaataaggcgc AAAGGTACCACTAAAGGAATTGCGAA TAATAATTTTTTCACTATGACTGT >For_HC1 2422 >Rev_HC2_OT 2435 gtgctaataaggcgcgccaattctat ACGCAGAGAACCGCCTGGCTGCACCA ttcaaggagacagtcatagtgaaa GCCCACCTCCGCTTTCCACCAGCT >For_HC2_OT 2423 >Rev_HC3_OT 2436 tttagtggtacctttctattctcact TTTCACGTTCACTGATTATACCATGG ccGAGGTTCAGCTGGTGGAAAGCG ATTGGGTTCGCCAGGCGCCGGGTA >For_HC3_OT 2424 >Rev_HC4_OT 2437 GGCGGTTCTCTGCGTCTGAGCTGCGC CCCTTAAAACGTTGGTTGTAAATTGA TGCCTCCGGTTTCACGTTCACTGA GCCACCAGAGTTAGGGTTTACGTC >For_HC4_OT 2425 >Rev_HC5_OT 2438 GCCAGGCGCCGGGTAAAGGCCTTGAA TTCTGCACGCAGCGAATTCATCTGTA TGGGTGGCCGACGTAAACCCTAAC AATAGAGTGTGTTTTTAGAGCGAT >For_HC5_OT 2426 >Rev_HC6_OT 2439 CCAACGTTTTAAGGGTCGTTTCACCC TGCCTTGGCCCCAATAGTCAAAGTAA TGAGCGTAGATCGCTCTAAAAACA AAGGACGGGCCCAGATTGCGTGCA >For_HC6_OT 2427 >Rev_HC7 2440 TCGCTGCCTGCAGAAGACACCGCTGT GATTTCGAGCTTGGGGCCAGCGGAAA TTATTACTGTGCACGCAATCTGGG CACTGACGGACCTTTAGTGCTTGC >For_HC7_OT 2428 >Rev_HCrescue_VH_OT 2441 ATTGGGGCCAAGGCACGTTGGTCACC GATTTCGAGCTTGGG GTGAGTAGCGCAAGCACTAAAGGT >Rev_HC7_OT_PCR 2429 >Rev_HC8 2442 ACCTTTAGTGCTTGCGCTACTCACGG GGAGACAGTGACCGGTTCCGGGAAGT TGACCAACGTGCCTTGGCCCCAAT AATCTTTCACCAGACAGCCCAGCG >For_HC8 2430 >Rev_HC9 2443 CCCAAGCTCGAAATCCACGTCCGGTG TATACAAGCCGCTAGACTGCAAAACC GCACCGCCGCGCTGGGCTGTCTGG GCAGGGAAAGTATGTACACCCGAG >For_HC9 2431 >Rev_HC10 2444 CCGGTCACTGTCTCCTGGAACTCGGG TGGTTCACATTGCAAATATACGTCTG TGCACTTACCTCGGGTGTACATAC GGTGCCCAGAGAGCTTGAAGGCAC >For_HC10 2432 >Rev_HC11 2445 CTAGCGGCTTGTATAGCCTGTCAAGC TTTTTGTTCTGCGGCCGCACAGCTCT GTTGTGACCGTGCCTTCAAGCTCT TCGGTTCCACTTTCTTATCCA >For_HC11 2433 >Rev_HCrescue 2446 TTGCAATGTGAACCACAAACCGAGTA TTTTTGTTCTGCGGC ACACCAAAGTGGATAAGAAAGTGG 

The invention claimed is:
 1. Isolated amino acid sequence that specifically binds HER2 and inhibits binding of trastuzumab to HER2, and that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: CDR1 is SEQ ID NO:462, and CDR2 is SEQ ID NO:1012, and CDR3 is SEQ ID NO:1562.
 2. The amino acid sequence according to claim 1, that is a domain antibody, a single domain antibody, a VHH, a humanized VHH or a camelized VH.
 3. The amino acid sequence according to claim 1, that essentially consists of a VHH, humanized VHH or camelized VH that has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22 or 2051-232, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded.
 4. The amino acid sequence according to claim 3, wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from: at position 11: L, M, S, V, W, at position 37: F, Y, H, I, L, V, at position 44: G, E, A, D, Q, R, S, L, at position 45: L, R, C, I, L, P, Q, V, at position 47: W, L, F, A, G, I, M, R, S, V, Y, at position 83: R, K, N, E, G, I, M, Q, T, at position 84: P, A, L, R, S, T, D, V, at position 103: W, P, R, S, at position 104: G, D, and at position 108: Q, L, R.
 5. The amino acid sequence according to claim 1, that is SEQ ID NO:2112.
 6. Isolated construct that comprises or essentially consists of one or more amino acid sequences according to claim 1, and further comprises one or more other groups, residues, moieties or binding units.
 7. Construct according to claim 6, which is a multiparatopic construct, comprising at least one amino acid sequence that binds a first epitope of HER-2 and at least one amino acid sequence that binds a second epitope of HER-2 different from the first epitope.
 8. Biparatopic construct according to claim 7, which can simultaneously bind the pertuzumab binding site on HER2 and to the trastuzumab binding site on HER2.
 9. Construct according to claim 6, wherein said construct specifically binds the trastuzumab binding site on HER2 and specifically binds the pertuzumab binding site on HER2.
 10. Construct according to claim 6, wherein said construct competes with trastuzumab and pertuzumab for binding to HER2 and/or wherein said construct inhibits binding of trastuzumab and pertuzumab to HER2.
 11. Construct according to claim 6, that comprises or consists of one of SEQ ID NO's: 2346 or
 2347. 12. Construct according to claim 6, which has an increased half-life, compared to the corresponding one or more amino acid sequences per se.
 13. Construct according to claim 6 that is selected from any of SEQ ID NO's: 2330 and
 2335. 14. Composition comprising at least one construct according to claim
 6. 15. Monovalent construct, comprising or essentially consisting of one amino acid sequence according to claim
 1. 16. Composition, comprising at least one amino acid sequence according to claim
 1. 